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Can CRISPR/Cas9 target multiple targets?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Updated 11/27/2015

 

 

CRISPR/Cas9 Gets a Boost from tRNA

04/07/2015

Nicholas Miliaras PhD

CRISPR/Cas9 has advanced genome editing and revolutionized molecular biology perhaps even more than the restriction enzyme. But can it edit multiple targets efficiently?

 

For CRISPR/Cas9 editing, single guide RNAs (sgRNAs) direct the bacterial Cas9 endonuclease to specific loci, allowing targeting of almost any gene. But is it possible to efficiently target multiple genes? “You can express one, two, or even three sgRNAs pretty easily, but if you want to do four, five, or more, it becomes difficult,” Yinong Yang at Pennsylvania State University said.

Yang’s team addressed this question in a Proceedings of the National Academy of Science paper by turning to the cell’s own tRNA processing systems. The group created polycistronic tRNA-gRNA (PTG) constructs that consisted of an sgRNA flanked by a pre-tRNA gene; the cell’s endogenous RNases can then cleave one or multiple transcribed gRNAs from the cistron to direct Cas9 to target genes.

 

Schematic depiction of the synthetic tRNA-gRNA gene. Credit: Yinong Yang.

 

“The beauty of this approach is that the 77 bp pre-tRNA gene contains internal promoter elements (box A and B) to recruit the RNA Pol III complex, so maybe you don’t even need a promoter. The Pol III promoter [which is currently used to drive expression of the sgRNA] isn’t very strong, so the tRNA will give you enhanced expression of multiple RNAs.”

The group first tested the PTG in rice protoplasts and soon realized that existing CRISPR/Cas9 vectors can be used to express PTGs. They also observed that the PTGs were more effective at introducing insertions or deletions than sgRNAs, perhaps owing to their higher expression levels from the endogenous tRNA enhancers.

Yang and his colleagues next asked if it was possible to introduce deletions in multiple genes by targeting the MAP kinase components MPK1, MPK2, MPK5, and MPK6 individually and in combinations of two or four. The PTG system introduced deletions for up to four genes, although there was a two-fold reduction in editing efficiency, which the authors attribute to competition for Cas9 among the multiple gRNAs. They then usedAgrobacterium-mediated transformation to transform mature rice plants with sgRNAs or PTGs for MPK genes and observed a higher mutational frequency of bi-allelic mutations and deletions in the plants transformed with the PTGs. Finally, they were able to target the phytoene desaturase (PDS) gene to generate a photo-bleached phenotype in the resulting plants. While they only obtained a single line carrying the fragment deletion of PDS, the mutational efficiency for PTGs was 100 percent.

 

Reference:

Xie, K, Minkenberg B, and Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112.

 

 

 

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system
Kabin Xie, Bastian Minkenberg, and Yinong Yang1
Department of Plant Pathology and Environmental Microbiology and the Huck Institutes of the Life Sciences,
Pennsylvania State University, University Park, PA 16802

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved February 3, 2015

The clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/ Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

CRISPR/Cas9 | tRNA processing | genome editing | multiplex

 

Significance The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has recently emerged as an efficient and versatile tool for genome editing in various organisms. However, its targeting capability and multiplex editing efficiency are often limited by the guide RNA (gRNA)-expressing device. This study demonstrates a general strategy and platform for precise processing and efficient production of numerous gRNAs in vivo from a synthetic polycistronic gene via the endogenous tRNA-processing system. This strategy is shown to significantly increase CRISPR/Cas9 multiplex editing capability and efficiency in plants and is expected to have broad applications for small RNA expression and genome engineering.

 

Fig. 1. Engineering the endogenous tRNA system for multiplex genome editing with CRISPR/Cas9. (A) The eukaryotic pre-tRNA with 5′ leader and 3′ trailer is cleaved by RNase P and RNase Z at specific sites. (B) Transcription of tRNA gene with RNA polymerase III (Pol III). The box A and box B elements in the tRNA gene function as internal transcriptional elements and are bound by transcription factor IIIC (TFIII C), which recruits TFIIIB and Pol III to start transcription. (C) Schematic depiction of the PTG/Cas9 method for simultaneously targeting multiple sites. The synthetic PTG consists of tandemly arrayed tRNA-gRNA units, with each gRNA containing a target-specific spacer (labeled as a diamond with different color) and conserved gRNA scaffold (rectangle). The tRNA containing box A and B elements is shown as round rectangles. The primary transcript of PTG is cleaved by endogenous RNase P and RNase Z (labeled as scissors) to release mature gRNAs and tRNA (red lines of cloverleaf structure). The excised mature gRNAs direct Cas9 to multiple targets.

 

Strategy to Engineer a tRNA-processing System for Producing Numerous gRNAs

Precise Processing of PTG to Produce Functional gRNAs with Desired Targeting Sequences

Fig. 2. Precise excision of functional gRNAs in vivo from synthetic PTG genes. (A) The architecture of two sgRNA genes and four PTGs to produce one gRNA. (B) Sequence and predicted secondary structure of tRNA–gRNA–tRNA fusion of PTG gene. The bases of the tRNA region are indicated with red color and the tRNA 5′ leader is shown in lowercase. The gRNA is indicated in black, and the gRNA spacer sequence is shown as N. (C–F) Examination of mature gRNAs produced from sgRNA or PTGs with cRT-PCR. Total RNAs from the protoplasts expressing empty vector were used as control (CK). Arrows indicate mature gRNAs amplified by cRT-PCR, and asterisks indicate the nonspecifically amplified rRNA. (G) Summary of excision sites in PTG according to mapped gRNA ends from cRT-PCR (SI Appendix, Figs. S3–S5). Arrows indicate the cleavage sites in PTG to release gRNA. The mature gRNA 5′ ends were excised from PTG exactly at the tRNA–gRNA fusion site in all cRT-PCR results whereas its 3′ ends shifted 1–4 nt within the tRNA 5′ leader (lowercase). (H) gRNA produced from U3p:sgRNA. All detected U3p:sgRNA-produced gRNA started with ribonucleotide A and terminated with multiple Us. (I) Introduction of indels at the desired sites by PTG1:Cas9 or PTG2:Cas9 in rice protoplasts as shown by PCR/RE. Arrows indicate mutated fragments resistant to RE digestion. The indel frequency is indicated at the bottom. (J) Relative expression of sgRNA1/2 and PTG1/2 in rice protoplasts. Data represent mean ± SD. ND, not detected. CK, empty vector control.

 

Efficient Multiplex Genome Editing in Rice Protoplasts via PTG/Cas9.

Fig. 3. Simultaneous editing of multiple genomic sites in rice protoplasts expressing PTG:Cas9. (A) Architecture, gRNA components, and targets of PTGs for multiplex genome editing. (B) PCR detection of chromosomal fragment deletion at targeted loci in rice protoplasts expressing respective PTGs with Cas9. Successful deletion is shown as truncated PCR product (indicated with arrows). The chromosomal fragment deletion frequency (del %) is indicated at the bottom of each lane. The protoplast samples expressing an empty vector were used as control (CK). (C) Representative sequences of chromosomal fragment deletion aligned with that of WT. The gRNA paired region is labeled with green color, and the PAM region is shown in red color letters. The number at the end indicates deleted (−) or inserted (+) bases between two Cas9 cuts. The total length between two Cas9 cut sites (labeled with scissor) is indicated on the top. Short lines in the aligned sequences indicate deletions.

 

Improving Multiplex Genome Editing in Stable Transgenic Plants with PTG/Cas9

Table 1. Targeted mutation efficiency in PTG:Cas9 vs. sgRNA:Cas9 plants

Fig. 4. Highly efficient targeted mutagenesis in transgenic rice expressing PTG:Cas9. (A and B) Chromosomal fragment deletion in PTG7:Cas9 plant at T0 generation. Of note, only mpk1 with 358-bp deletion (Δ358) was detected in genomic DNA. Sequence analysis of the PCR products (the number in parentheses) reveals an identical deletion pattern in the transgenic plant. (C) Albino seedlings were regenerated from calli transformed with PTG10:Cas9. Most T0 seedlings (87%, n = 15) exhibited a similar photo-bleach phenotype, suggesting a very high efficiency of knocking out PDS with PTG10:Cas9. Vec, control plants transformed with empty vector. (Scale bar: 5 cm.)

 

We developed a general strategy and platform to produce multiple gRNAs from a single synthetic PTG gene by hijacking the endogenous tRNA-processing system (Fig. 1). We also provided a framework to design, synthesize, and use PTG for multiplex genome editing with Cas9. These PTGs were expressed with Pol III promoters (e.g., U3p) in the same manner as sgRNA genes but were not obligated to start with a specific nucleotide (Fig. 2). As a result, current CRISPR/Cas9 vectors for expressing sgRNA could be directly used to express PTG for multiplex genome editing as we demonstrated in this study.

By producing multiple gRNAs from a single polycistronic gene, the PTG technology could be used to improve simultaneous mutagenesis of multiple genomic loci or deletion of short chromosomal fragments (Figs. 3 and 4). Such a genome engineering approach may lead to simultaneous knock-out of multiple protein coding genes or deletion of noncoding RNA regions and other genetic elements. In addition to targeted mutagenesis/ deletion, the PTG approach could facilitate other Cas9-based applications in which multiple gRNAs are required. For example, PTG could be used with Cas9 nickase to improve targeting fidelity (13, 33, 34), or with deactivated Cas9 transcriptionalactivator or -repressor to manipulate multiple gene expression (35, 36). Given the high processing accuracy and capability of RNase P and RNase Z that we observed (Fig. 2), the tRNAprocessing system also could be used as a general platform to produce other regulatory RNAs (e.g., short hairpin RNA or artificial microRNA) from a single synthetic gene. These different classes of regulatory RNAs, like gRNA and short hairpin RNA, also could be compacted into a single polycistronic gene to develop more sophisticated devices for genetic engineering.

Recently, polycistronic transcripts that fused gRNA with a 28-nt RNA (referred to as gRNA-28nt) were successfully used to guide Cas9 to target up to four targets in human cells (12, 13). The synthetic gene with a gRNA-28nt architecture produced mature gRNAs with a 28-nt extra 3′ sequence and also required coexpressing the endonuclease Csy4 from Pseudomonas aeruginosa to cleave the transcript. In comparison with the gRNA-28nt strategy, our approach uses a robust endogenous tRNA-processing system that enables precise production of gRNAs with only a 1- to 4-nt extra sequence at the gRNA 3′ end (Figs. 1 and 2) and carries no additional risk of endonuclease Csy4 toxicity to recipients. Given the extremely large number of tRNA genes with variable sequences and the fact that RNase P and RNase Z precisely recognize RNA substrates with tRNA-like structures (18, 37), there are many choices of tRNA sequences to be embedded in PTG. Furthermore, the tRNA-processing system is universal in all living organisms; thus, the PTG technology could be directly adapted to other organisms for Cas9-mediated genome engineering.

When multiple double-strand breaks (DSBs) in genomic DNA were generated by PTG/Cas9 in rice plants, indels resulting from error-prone NHEJ repairing occurred more frequently than fragment deletions generated by directly joining two DSBs (SI Appendix, Figs. S10 and S11). To date, the molecular mechanism by which two DSBs directly link together to generate chromosomal translocation or fragment deletion in vivo is largely unclear. We speculate that the process leading to such a chromosomal disorder may require two DSBs at the same time interval and is likely determined by the highly dynamic interaction between gRNA/Cas9 cutting and endogenous DNA repairing and also by the distance between DSBs. Due to the differences in the delivery, expression, and activity of gRNAs and Cas9, it is not surprising to see some discrepancies in fragment-deletion frequency between protoplasts (Fig. 3B) and stable transgenic plants and among different PTG transgenic lines (Fig. 4A and SI Appendix, Figs. S9–S11). Because the PTG technology enables the generation of many DSBs in genomic DNAs, it may provide an efficient tool to help dissect the molecular process of chromosomal deletion. More importantly, the PTG technology significantly improves multiplex editing capability and efficiency and is expected to facilitate more sophisticated Cas9 applications, such as targeted mutagenesis and deletion of redundant genes or Fig. 4. Highly efficient targeted mutagenesis in transgenic rice expressing PTG:Cas9. (A and B) Chromosomal fragment deletion in PTG7:Cas9 plant at T0 generation. Of note, only mpk1 with 358-bp deletion (Δ358) was detected in genomic DNA. Sequence analysis of the PCR products (the number in parentheses) reveals an identical deletion pattern in the transgenic plant. (C) Albino seedlings were regenerated from calli transformed with PTG10:Cas9. Most T0 seedlings (87%, n = 15) exhibited a similar photo-bleach phenotype, suggesting a very high efficiency of knocking out PDS with PTG10:Cas9. Vec, control plants transformed with empty vector. (Scale bar: 5 cm.) genetic elements, transcriptional modulation of multiple genes and pathways, modification and labeling of numerous genomic sites, site-specific integration, and gene replacement.

3570-3575 | www.pnas.org/cgi/doi/10.1073/pnas.1420294112 Xie et al. genetic elements, transcriptional modulation of multiple genes and pathways, modification and labeling of numerous genomic sites, site-specific integration, and gene replacement

 

Validating “predicted” regulatory elements through CRISPR editing of the non-coding genome

CRISPR Cas9 genome editing of the non-coding genome

CRISPR/Cas9-mediated genome editing is not only an efficient way to create gene KO & KI, but is a uniquely powerful tool to functionally characterize the >98% of the genome that does not encode protein. A new study demonstrates how CRISPR can be used to systematically validate putative regulatory elements described by the ENCODE and EPIGENOME projects: even in a repeat-rich genomic region, a genomic insulator upstream of mouse tyrosinase was efficiently deleted or inverted, with no significant off-target effects and high efficiency in vivo, demonstrating a functional role for this noncoding region in regulating tyrosinase gene expression and mouse coat pigmentation.

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Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis.
Seruggia D1,2Almudena Fernández1,2Marta Cantero1,2Pawel Pelczar3 and Lluis Montoliu1,2,*
Nucleic Acids Res. 2015 May 26;43(10):4855-67. Read the Free Full Text

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

 

Non-coding DNA regulatory elements are composed of arrays of DNA–protein binding sites extending over tens to hundreds of base pairs that are occupied by multiple groups of transcription factors. DNA methylation, covalent modification of histone proteins and DNase I hypersensitivity profiles allow unbiased identification of such elements as regions of active chromatin that might be relevant in the regulation of different genes in a particular tissue or condition. Systematic ChIP-Sequencing (chromatin immunoprecipitation coupled with massive parallel sequencing) using antibodies specific for a variety of nuclear factors, applied to several human cell lines (1) and mouse tissues (2), served to identify cell type-specific regulatory elements accounting for almost 80% of the non-coding fraction of the genome. These studies, globally known as the ENCODE project (Encyclopaedia of DNA Elements; (3)) underline the rich proportion of functional elements existing within the non-coding areas of mammalian genomes. The recent publication of the human EPIGENOME project has provided additional evidence for the relevance of DNA regulatory elements in controlling gene expression (4). However, many functional experiments are required to unequivocally demonstrate the links between the observed biochemical chromatin features and the predicted biological function (5).

In the past years, the relevance of non-coding regions has been typically addressed, in vivo, using genomic-type transgenes (mostly bacterial and yeast artificial chromosomes, BACs and YACs; reviewed in (6)) carrying the inactivation of putative regulatory elements surrounded by tens to hundreds of kilo bases of genomic sequences of a suitable endogenous gene or coupled to a reporter gene (711). In this manner, large genomic fragments have been easily manipulated using homologous recombination in bacteria (12) and yeast (13) and then introduced into the mouse germline by standard procedures (1415). However, variability is often observed between transgenic lines generated with BAC- or YAC-type transgenes, suggesting that position effects can influence transgene expression, even on large constructs (1521). In addition, not all loci fit in such artificial chromosome-type transgenes, for example, large multi-gene syntenic blocks or gene clusters, whose transcriptional regulation programs during development are coordinated (22).

 

Here, we propose a simple strategy to functionally validate the relevance of non-coding regulatory elements in the mouse genome, in vivo. We have applied CRISPR–Cas9-mediated mutagenesis tools to inactivate, via deletion, a key regulatory sequence identified in the mouse Tyr gene (48).

We previously reported a DNAse hypersensitive (HS) site, located at ∼12 kb 5′-upstream of the mouse Tyr transcription start site (TSS), associated with a melanocyte-specific enhancer that was required for the correct expression of the Tyr gene (39). The deletion or inactivation of this element, in the context of YAC transgenesis, produced mice displaying variegation with severely reduced coat color pigmentation, supporting the notion that this key element was acting as a Locus Control Region (LCR) (7)). Homologous sequences to this mouse Tyr 5′ element were also found within the 5′ end of the human TYR locus, suggesting that mutations in this element could also impair the function of the human TYR gene (54). Traditional molecular diagnosis efforts for OCA1 patients regularly fail to detect all TYR mutations, beyond coding, promoter and limited intronic DNA sequences routinely explored. Consequently, it has been repeatedly suggested that mutations in non-coding regions could be responsible for some of these unknown non-functional TYR alleles (38,55,56). Interestingly, the recent human epigenome data released for many cellular types, including skin melanocytes, describes a regulatory element (a DNAse HS) located at ∼10 kb 5′ upstream of the human TYR gene promoter ((4); Supplementary Figure S8) at the same genomic location as was previously predicted (54). Until now, the direct relevance of TYR or Tyrregulatory elements could not be adequately studied at the endogenous loci. Instead, their role had to be inferred from results obtained using diverse standard and chromosome-type transgenes in mice (17,35).

Further studies revealed that the Tyr LCR had properties typical of genomic boundaries or insulators (57), including the capacity of establishing barriers that prevent spreading of heterochromatin and epigenetic silencing (29), and enhancer-blocking activity (40). The function of insulators is rather complex and strictly dependent on the interactions with other proximal and distal sequences in the genomic locus (43,5860). The context-dependent activity of insulators should be therefore characterized in their native chromosomal context by gene targeting. However, the presence of repetitive sequences surrounding theTyr 5′ boundary element (29) invalidated the application of standard gene targeting approaches. As an alternative, we decided to use CRISPR–Cas9-mediated mutagenesis to overcome the limitations of classical gene targeting strategies.

Similar approaches have been recently reported to address the role of a distal Sox2 enhancer in mouse ES cells (5). Endonuclease-mediated deletions, using Transcription Activator-Like Effector Nucleases (TALENs) and Zinc-Finger Nucleases (ZFN), have been described in zebrafish (61). CRISPR–Cas9 was also used to characterize mutations found at the distal enhancer of the TAL1 oncogene in human tumor cell lines (62). Additionally, mouse models were generated using CRISPR–Cas9 in mouse ES cells to reproduce structural variants, including deletions and inversions, found in human disease (63).

In this work, we report that defined deletions and inversions in non-coding regions can be efficiently generated in vivo by CRISPR–Cas9 approaches using sgRNAs directed to adjacent genomic target sites. CRISPR–Cas9 RNA species are injected into fertilized eggs where they generate mutations at the target sequences. These mutations are then efficiently transmitted through the germ line. Using this strategy, mouse embryos are exposed to a limited amount of Cas9 nuclease for a short time, thus minimizing the risk of off-target mutations. Indeed, in our screen, no undesired mutations were detected at the six genomic loci highly similar to the targeted sequences under investigation. In contrast to this, approaches based on the delivery of CRISPR–Cas9 plasmids to somatic or ES cells may increase the associated risk of off-target mutations since exposure to the Cas9 nuclease is massive and prolonged (31).

Inactivation of the Tyr 5′ boundary element in genomic-type transgenes resulted in a severe reduction in coat color pigmentation, pointing to a relevant role for this non-coding sequence (7). However, these results were based on ectopic chromosomal locations, where variables such as transgene integrity, copy number and integration site could affect the overall gene expression program (1521). Because of this, our vision was to target this 5′ boundary element directly at the Tyr endogenous locus, where we could unequivocally link this element to the observed phenotype without further uncontrolled variables. In actual fact, a comparative analysis of Tyr expression patterns in YAC Tyr transgenic mouse lines and TYRINS5 edited lines reveals fundamental differences in both melanocytes and RPE cells (Figures 4A, C, D, 5A, B and C). Deleting the Tyr 5′ boundary appears to have a milder effect in skin and choroidal melanocytes and a more limited impact in RPE cells, suggesting that additional regulatory elements may be responsible for controlling Tyr gene expression in RPE cells. Indeed, the presence of RPE-specific regulatory elements further upstream had been previously proposed and investigated in mice using BAC Tyr transgenes engineered with a lacZ reporter gene and variable combinations of Tyr 5′ genomic sequences (64).

 

CRISPR genome editing in human cells: improved targeting with the H1 promoter

A recent paper in Nature Communications reports success with a clever technique to make CRISPR-mediated genome editing easier in human cells. Compared to the commonly-used U6 promoter, driving guide RNA expression from the H1 promoter more than doubles the number of targetable sites within the genomes of humans and other eukaryotes.

Why is H1 more versatile than U6? The U6 promoter initiates transcription from a guanosine (G) nucleotide, while the H1 promoter can initiate transcription from A or G. In designing a gRNA sequence, the requirement for the protospacer adjacent motif (PAM) sequence “NGG” at the end of a 20-mer means that U6-driven gRNA must fit the pattern GN19NGG. But H1-driven gRNAs can also target sequences of the form AN19NGG, which occur 15% more frequently than GN19NGG within the human genome.

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Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.
Vinod RanganathanKarl WahlinJulien Maruotti & Donald J. Zack  
Nat Commun. 2014 Aug 8;5:4516. Read Full Text
http://dx.doi.org:/10.1038/ncomms5516

The repurposed CRISPR–​Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

 

Figure 1: Evaluating the ability to direct CRISPR targeting via gRNA synthesis from the H1 promoter.

Evaluating the ability to direct CRISPR targeting via gRNA synthesis from the H1 promoter.

(a) Schematic illustration depicting the gRNA expression constructs. Above, the U6 promoter only expresses gRNAs with a +1 guanosine nucleotide; below, the H1 promoter can drive expression of gRNAs initiating at either purine (adenosine…

Figure 2: Bioinformatics analysis of GN19NGG and AN19NGG sites in the genome.

Bioinformatics analysis of GN19NGG and AN19NGG sites in the genome.

(a) Circos plot depicting the frequency of CRISPR sites in the human genome. The outside circle depicts the human chromosome ideograms. Moving inwards, GN19NGG (orange), AN19NGG (blue) and RN19NGG (purple) CRISPR sites frequency is indi…

 

Could CRISPR technology be used to cure AIDS and other devastating viral diseases?

Why are viral diseases like AIDS still incurable? Although antiretroviral drugs can effectively control viral load in many patients, the permanent integration of viral DNA into a host genome means that patients remain vulnerable to re-activation of a latent virus. Exciting new research now shows that CRISPR technology can remove HIV DNA that has integrated into the host genome in human cells, re-igniting our hopes for developing a true cure for AIDS.

CRISPR-mediated genome editing is revolutionizing biomedical research due to its precise targeting, high efficiency, and ease of use in any cell type or experimental system. CRISPR has been used to create new transgenic animal models for basic and translational research, and it holds promise for use in gene therapy and other medical applications.

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Read the full publication: RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection.
Wenhui Hua,1,2Rafal Kaminskia,1Fan YangaYonggang ZhangaLaura CosentinoaFang LiaBiao Luob, et al. 
Proc Natl Acad Sci U S A. 2014 Jul 21; 111(31):11461–11466
http://dx.doi.org:/10.1073/pnas.1405186111

Significance

For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

 

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

 

Infection with HIV-1 is a major public health problem affecting more than 35 million people worldwide (1). Current therapy for controlling HIV-1 infection and impeding AIDS development (highly active antiretroviral therapy; HAART) includes a mixture of compounds that suppress various steps of the viral life cycle (2). HAART profoundly reduces viral replication in cells that support HIV-1 infection and reduces plasma viremia to a minimal level but neither suppresses low-level viral genome expression and replication in tissues nor targets the latently infected cells that serve as a reservoir for HIV-1, including brain macrophages, microglia, and astrocytes, gut-associated lymphoid cells, and others (3, 4). HIV-1 persists in ∼106 cells per patient during HAART, and is linked to comorbidities including heart and renal diseases, osteopenia, and neurological disorders (5). Because current therapies are unable to suppress viral gene transcription from integrated proviral DNA or eliminate the transcriptionally silent proviral genomes, low-level viral protein production by latently infected cells may contribute to multiple illnesses in the aging HIV-1–infected patient population. Supporting this notion, pathogenic viral proteins including transactivator of transcription (Tat) are present in the cerebrospinal fluid of HIV-1–positive patients receiving HAART (6). To prevent viral protein expression and viral reactivation in latently infected host cells, new strategies are thus needed to permanently disable the HIV-1 genome by eradicating large segments of integrated proviral DNA.

Advances in the engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) that can disrupt target genes have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome (710). These approaches have been used to disrupt HIV-1 entry coreceptors C-C chemokine receptor 5 (CCR5) or C-C-C chemokine receptor 4 (CXCR4) and proviral DNA-encoding viral proteins (8, 9). CCR5 gene-targeting ZFNs are in phase II clinical trials for HIV-1/AIDS treatment (11). Also, various gene editing technologies have recently been shown to remove the proviral HIV-1 DNA from the host cell genome by targeting its highly conserved 5′ and 3′ long terminal repeats (LTRs) (12, 13). However, introduction of nucleases into cells via these nuclease-based genomic editing approaches remains inefficient and partially selective to remove the entire HIV-1 genome. Thus, the key barrier to their clinical translation is insufficient gene specificity to prevent potential off-target effects (toxicities). To achieve highly specific HIV-1 genome editing, we combined approaches to identify HIV-1 targets while circumventing host off-target effects. The resulting highly specific Cas9-based method proved capable of eradicating integrated HIV-1 DNA with high efficiency from latently infected human “reservoir” cell types, and prevented their infection by HIV-1.

 

Here, we found that LTR-directed gRNA/Cas9 eradicates the HIV-1 genome and effectively immunizes target cells against HIV-1 reactivation and infection with high specificity and efficiency. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses. In the current study, we have mainly focused our efforts on myeloid lineage cells (microglia/macrophage), which are the primary cell types that harbor HIV-1 in the brain. However, this proof of concept is certainly applicable to any other cell type, including T-lymphoid cells (Fig. S6) (12, 13), astrocytes, and neural stem cells.

Our combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus. In addition to an extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, the key design attributes in our study included: bioinformatic screening using the strictest 12-bp+NGG target selection criteria to exclude off-target human transcriptome or (even rarely) untranslated genomic sites; avoiding transcription factor binding sites within the HIV-1 LTR promoter (potentially conserved in the host genome); selection of LTR-A- and -B-directed, 30-bp protospacer and also precrRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp protospacer-based, chimeric crRNA-tracRNA system (16, 30); and WGS, Sanger sequencing, and SURVEYOR assay, to identify and exclude potential off-target effects. Indeed, the use of newly developed Cas9 double-nicking (23) and RNA-guided FokI nuclease (31, 32) may further assist identification of new targets within the various conserved regions of HIV-1 with reduced off-target effects.

More recently, a clinical trial using the ZFN gene editing strategy was launched to disrupt the gene encoding the HIV-1 coreceptor, CCR5 (8, 9, 11). Functional knockout of CCR5 in autologous CD4 T cells of a small cohort of patients revealed that in one out of four enrolled subjects, the viral load remained undetectable at the time of treatment (33). Similarly, TALEN and Cas9 have been tested experimentally for efficient disruption of CCR5 and CXCR4 (9, 28, 3437); therefore, taking them into consideration for clinical trials is anticipated. Whether or not the strategies targeting HIV-1 entry can reach the “sterile” cure of AIDS remains to be seen. Our results show that the HIV-1 Cas9/gRNA system has the ability to target more than one copy of the LTR, which are positioned on different chromosomes, suggesting that this genome-editing system can alter the DNA sequence of HIV-1 in latently infected patient’s cells harboring multiple proviral DNAs. To further ensure high editing efficacy and consistency of our technology, one may consider the most stable region of HIV-1 genome as a target to eradicate HIV-1 in patient samples, which may not harbor only one strain of HIV-1. Alternatively, one may develop personalized treatment modalities based on the data from deep sequencing of the patient-derived viral genome before engineering therapeutic Cas9/gRNA molecules.

Our results also demonstrate, for the first time to our knowledge, that Cas9/gRNA genome editing can be used to immunize cells against HIV-1 infection. The preventative vaccination is independent of HIV-1 strain’s diversity because the system targets genomic sequences regardless of how the viruses enter the infected cells. Interestingly, the preexistence of the Cas9/gRNA system in cells leads to a rapid elimination of the new HIV-1 before it integrates into the host genome, just like the way by which the bacteria defense system evolved to combat phage infection (38). Similarly, a gene-editing-based vaccine strategy may be effective in eradicating postintegrated HIV-1 genome and newly packaged proviruses in cells. Therefore, investigation of such HIV-1 vaccination in various latent reservoir cells and animal models with stable expression of Cas9/LTR-gRNAs presents an important next step to assess the ability of Cas9 to eradicate viral reservoirs in vivo. Moreover, in light of recent data illustrating efficient in vitro genome editing using a mixture of Cas9/gRNA and DNA (3942), one may explore various systems for delivery of Cas9/LTR-gRNA via various routes for immunizing high-risk subjects. Once advanced, one may use gene therapies (viral vector and nanoparticle) and transplantation of autologous Cas9/gRNA-modified bone marrow stem/progenitor cells (43, 44) or inducible pluripotent stem cells for eradicating HIV-1 infection.

Here, we demonstrated the high specificity of Cas9/gRNAs in editing HIV-1 target genome. Results from subclone data revealed the strict dependence of genome editing on the presence of both Cas9 and gRNA. Moreover, only one nucleotide mismatch in the designed gRNA target will disable the editing potency. In addition, all four of our designed LTR gRNAs worked well with different cell lines, indicating that the editing is more efficient in the HIV-1 genome than the host cellular genome, wherein not all designed gRNAs are functional, which may be due to different epigenetic regulation, variable genome accessibility, or other reasons. Given the ease and rapidity of Cas9/gRNA development, even if HIV-1 mutations confer resistance to one Cas9/gRNA-based therapy, as described above, HIV-1 variants can be genotyped to enable another personalized therapy for individual patients (10).

 

CRISPR-Cas9 Gene Editing: Check Three Times, Cut Once

http://www.technologynetworks.com/Genomics/news.aspx?ID=185167

Two new studies from UC Berkeley should give scientists who use CRISPR-Cas9 for genome engineering greater confidence that they won’t inadvertently edit the wrong DNA.

The gene editing technique, created by UC Berkeley biochemist Jennifer Doudna and her colleague, Emmanuelle Charpentier, director of the Max Planck Institute of Infection Biology in Berlin, has taken the research and clinical communities by storm as an easy and cheap way to make precise changes in DNA in order to disable genes, correct genetic disorders or insert mutated genes into animals to create models of human disease.

The two new reports from Doudna’s lab and that of UC Berkeley colleague Robert Tjian show in much greater detail how the Cas9 protein searches through billions of base pairs in a cell to find the right DNA sequence, and how Cas9 determines whether to bind, or bind and cut, thereby initiating gene editing. Based on these experiments, Cas9 appears to have at least three ways of checking to make sure it finds the right target DNA before it takes the irrevocable step of making a cut.

“CRISPR-Cas9 has evolved for accurate DNA targeting, and we now understand the molecular basis for its seek-and-cleave activity, which helps limit off-target DNA editing,” said Doudna, a Howard Hughes Medical Institute investigator at UC Berkeley and professor of molecular and cell biology and of chemistry. Tjian is president of the Howard Hughes Medical Institute and a UC Berkeley professor of molecular and cell biology.

The studies also illustrate how well CRISPR/Cas9 works in human and animal cells – eukaryotes – even though “the technique was invented by bacteria to protect themselves from getting the flu,” Doudna said.

CRISPR-Cas9 is a hybrid of protein and RNA – the cousin to DNA – that functions as an efficient search-and-snip system in bacteria. It arose as a way to recognize and kill viruses, but Doudna and Charpentier realized that it could also work well in other cells, including humans, to facilitate genome editing. The Cas9 protein, obtained from the bacteria Streptococcus pyogenes, functions together with a “guide” RNA that targets a complementary 20-nucleotide stretch of DNA. Once the RNA identifies a sequence matching these nucleotides, Cas9 cuts the double-stranded DNA helix.

One study tracked Cas9-RNA molecules though the nucleus of mammalian cells as they rapidly searched through the entire genome to find and bind just the region targeted and no other.

“It’s crazy that the Cas9 complex manages to scan the vast space of eukaryotic genomes,” said graduate student Spencer Knight, first author of the paper.

Previous studies had suggested that there are many similar-looking DNA regions that Cas9 could bind and cut, which could limit its usefulness if precision were important. These off-target regions might share as few as four or five nucleotides with the 20-nucleotide primer, just enough for Cas9 to recognize.

“There is a lot of off-target binding by Cas9, but we found that these interactions are very brief – from milliseconds to seconds – before Cas9 moves on,” he said.

Because these exploratory bindings – perhaps as many as 300,000 of them – are often very short-lived, a few thousand CRISPR-Cas9 complexes can scour the entire genome to find one targeted stretch of DNA. Cas9 must also recognize a short three-base-pair DNA sequence immediately following the primer sequence, dubbed PAM, which occurs about 300 million times within the human genome.

“If Cas9 bound for tens of seconds or minutes at each off-target site, it would never, ever be able to find a target and cut in a timely manner,” Knight said.

Cas9’s final checkpoint

The other study, published online Oct. 28 in Nature, showed that once Cas9 binds to a region of DNA, it performs another check before two distant sections of the Cas9 protein complex come together, like the blades of a scissors, to precisely align the active sites that cut double-stranded DNA.

“We found that RNA-guided Cas9 can bind some off-target DNA sequences, which differ from the correct target by just a few mutations, very tightly. Surprisingly, though, the region of Cas9 that does the cutting is inhibited because of the imperfect match. But when the correctly matching DNA is located, Cas9 undergoes a large structural change that releases this inhibition and triggers DNA cutting,” said first author Samuel Sternberg, who recently received his Ph.D. at UC Berkeley. He was able to observe these changes using a fluorescently labeled version of the Cas9 complex.

“We think that this structural change is the last checkpoint, or proofreading stage, of the DNA targeting reaction,” he said. “First, Cas9 recognizes a short DNA segment next to the target – the PAM – then the target DNA is matched up with the guide RNA via Watson-Crick base-pairing. Finally, when a perfect match is identified, the last part of the protein swings into place to enable cutting and initiate genome editing.”

A smaller Cas9 protein from a different species of bacteria, Staphylococcus aureus, likely exploits the same strategy to improve the precision of DNA targeting, suggesting that “this important feature has been preserved throughout evolutionary time,” he added.

“This is good news, in that it suggests that you have more than one checkpoint to ensure correct Cas9 binding,” Knight said. “There’s not just sequence regulation, there is also temporal regulation: it has to engage with the DNA and park long enough that it can actually rearrange and cut.”

The discoveries from Doudna, Tjian and their teams shed light on the molecular basis of off-target effects during genome editing applications, and may guide the future design of more accurate Cas9 variants.

 

 

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Introduction to Protein Synthesis and Degradation

Curator: Larry H. Bernstein, MD, FCAP

 

 

Introduction to Protein Synthesis and Degradation

This chapter I made to follow signaling, rather than to precede it. I had already written much of the content before reorganizing the contents. The previous chapters on carbohydrate and on lipid metabolism have already provided much material on proteins and protein function, which was persuasive of the need to introduce signaling, which entails a substantial introduction to conformational changes in proteins that direct the trafficking of metabolic pathways, but more subtly uncovers an important role for microRNAs, not divorced from transcription, but involved in a non-transcriptional role.  This is where the classic model of molecular biology lacked any integration with emerging metabolic concepts concerning regulation. Consequently, the science was bereft of understanding the ties between the multiple convergence of transcripts, the selective inhibition of transcriptions, and the relative balance of aerobic and anaerobic metabolism, the weight of the pentose phosphate shunt, and the utilization of available energy source for synthetic and catabolic adaptive responses.

The first subchapter serves to introduce the importance of transcription in translational science.  The several subtitles that follow are intended to lay out the scope of the transcriptional activity, and also to direct attention toward the huge role of proteomics in the cell construct.  As we have already seen, proteins engage with carbohydrates and with lipids in important structural and signaling processes.  They are integrasl to the composition of the cytoskeleton, and also to the extracellular matrix.  Many proteins are actually enzymes, carrying out the transformation of some substrate, a derivative of the food we ingest.  They have a catalytic site, and they function with a cofactor – either a multivalent metal or a nucleotide.

The amino acids that go into protein synthesis include “indispensable” nutrients that are not made for use, but must be derived from animal protein, although the need is partially satisfied by plant sources. The essential amino acids are classified into well established groups. There are 20 amino acids commonly found in proteins.  They are classified into the following groups based on the chemical and/or structural properties of their side chains :

  1. Aliphatic Amino Acids
  2. Cyclic Amino Acid
  3. AAs with Hydroxyl or Sulfur-containing side chains
  4. Aromatic Amino Acids
  5. Basic Amino Acids
  6. Acidic Amino Acids and their Amides

Examples include:

Alanine                  aliphatic hydrophobic neutral
Arginine                 polar hydrophilic charged (+)
Cysteine                polar hydrophobic neutral
Glutamine             polar hydrophilic neutral
Histidine                aromatic polar hydrophilic charged (+)
Lysine                   polar hydrophilic charged (+)
Methionine            hydrophobic neutral
Serine                   polar hydrophilic neutral
Tyrosine                aromatic polar hydrophobic

Transcribe and Translate a Gene

  1. For each RNA base there is a corresponding DNA base
  2. Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein.
  3. mRNA is produced in the nucleus, and is transferred to the ribosome
  4. mRNA uses uracil instead of thymine
  5. the ribosome reads the RNA sequence and makes protein
  6. There is a sequence combination to fit each amino acid to a three letter RNA code
  7. The ribosome starts at AUG (start), and it reads each codon three letters at a time
  8. Stop codons are UAA, UAG and UGA

 

protein synthesis

protein synthesis

http://learn.genetics.utah.edu/content/molecules/transcribe/images/TandT.png

mcell-transcription-translation

mcell-transcription-translation

http://www.vcbio.science.ru.nl/images/cellcycle/mcell-transcription-translation_eng_zoom.gif

transcription_translation

transcription_translation

 

http://www.biologycorner.com/resources/transcription_translation.JPG

 

What about the purine inosine?

Inosine triphosphate pyrophosphatase – Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2′-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5′-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions.

Gastroenterology. 2011 Apr;140(4):1314-21.  http://dx.doi.org:/10.1053/j.gastro.2010.12.038. Epub 2011 Jan 1.

Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function.

Hitomi Y1, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB.

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown.

Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR).

ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

Ther Drug Monit. 2012 Aug;34(4):477-80.  http://dx.doi.org:/10.1097/FTD.0b013e31825c2703.

Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.

Citterio-Quentin A1, Salvi JP, Boulieu R.

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.

The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.

The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

 

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3

PeerJ 2:e270; http://dx.doi.org:/10.7717/peerj.270

Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher.We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression.We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 D 0.13). Based on this, our second strategy suggests that mRNA levels explain 81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimat that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the ab-sence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the 40% of genes that are non-expressed.

 

Related studies that reveal issues that are not part of this chapter:

  1. Ubiquitylation in relationship to tissue remodeling
  2. Post-translational modification of proteins
    1. Glycosylation
    2. Phosphorylation
    3. Methylation
    4. Nitrosylation
    5. Sulfation – sulfotransferases
      cell-matrix communication
    6. Acetylation and histone deacetylation (HDAC)
      Connecting Protein Phosphatase to 1α (PP1α)
      Acetylation complexes (such as CBP/p300 and PCAF)
      Sirtuins
      Rel/NF-kB Signal Transduction
      Homologous Recombination Pathway of Double-Strand DNA Repair
    7. Glycination
    8. cyclin dependent kinases (CDKs)
    9. lyase
    10. transferase

 

This year, the Lasker award for basic medical research went to Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) for their “discoveries concerning the unfolded protein response (UPR) — an intracellular quality control system that

detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.”

About UPR: Approximately a third of cellular proteins pass through the Endoplasmic Reticulum (ER) which performs stringent quality control of these proteins. All proteins need to assume the proper 3-dimensional shape in order to function properly in the harsh cellular environment. Related to this is the fact that cells are under constant stress and have to make rapid, real time decisions about survival or death.

A major indicator of stress is the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), which triggers a transcriptional cascade in order to increase the folding capacity of the ER. If the metabolic burden is too great and homeostasis cannot be achieved, the response shifts from

damage control to the induction of pro-apoptotic pathways that would ultimately cause cell death.

This response to unfolded proteins or the UPR is conserved among all eukaryotes, and dysfunction in this pathway underlies many human diseases, including Alzheimer’s, Parkinson’s, Diabetes and Cancer.

 

The discovery of a new class of human proteins with previously unidentified activities

In a landmark study conducted by scientists at the Scripps Research Institute, The Hong Kong University of Science and Technology, aTyr Pharma and their collaborators, a new class of human proteins has been discovered. These proteins [nearly 250], called Physiocrines belong to the aminoacyl tRNA synthetase gene family and carry out novel, diverse and distinct biological functions.

The aminoacyl tRNA synthetase gene family codes for a group of 20 ubiquitous enzymes almost all of which are part of the protein synthesis machinery. Using recombinant protein purification, deep sequencing technique, mass spectroscopy and cell based assays, the team made this discovery. The finding is significant, also because it highlights the alternate use of a gene family whose protein product normally performs catalytic activities for non-catalytic regulation of basic and complex physiological processes spanning metabolism, vascularization, stem cell biology and immunology

 

Muscle maintenance and regeneration – key player identified

Muscle tissue suffers from atrophy with age and its regenerative capacity also declines over time. Most molecules discovered thus far to boost tissue regeneration are also implicated in cancers.  During a quest to find safer alternatives that can regenerate tissue, scientists reported that the hormone Oxytocin is required for proper muscle tissue regeneration and homeostasis and that its levels decline with age.

Oxytocin could be an alternative to hormone replacement therapy as a way to combat aging and other organ related degeneration.

Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration (June 2014)

 

Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14289-94.   http://dx.doi.org:/10.1073/pnas.1407640111. Epub 2014 Sep 15.

Role of forkhead box protein A3 in age-associated metabolic decline.

Ma X1, Xu L1, Gavrilova O2, Mueller E3.

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter.

 

Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

RJ Weatheritt, TJ Gibson & MM Babu
Nature Structural & Molecular Biology 24 Aug, 2014; 21: 833–839 http://dx.do.orgi:/10.1038/nsmb.2876

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.

 

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.

 

An overview of the potential advantages conferred by distal-site protein synthesis

An overview of the potential advantages conferred by distal-site protein synthesis

 

Turquoise and red filled circle represents off-target and correct interaction partners, respectively. Wavy lines represent a disordered region within a distal site synthesis protein. Grey and red line in graphs represents profiles of t…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

 

Tweaking transcriptional programming for high quality recombinant protein production

Since overexpression of recombinant proteins in E. coli often leads to the formation of inclusion bodies, producing properly folded, soluble proteins is undoubtedly the most important end goal in a protein expression campaign. Various approaches have been devised to bypass the insolubility issues during E. coli expression and in a recent report a group of researchers discuss reprogramming the E. coli proteostasis [protein homeostasis] network to achieve high yields of soluble, functional protein. The premise of their studies is that the basal E. coli proteostasis network is insufficient, and often unable, to fold overexpressed proteins, thus clogging the folding machinery.

By overexpressing a mutant, negative-feedback deficient heat shock transcription factor [σ32 I54N] before and during overexpression of the protein of interest, reprogramming can be achieved, resulting in high yields of soluble and functional recombinant target protein. The authors explain that this method is better than simply co-expressing/over-expressing chaperones, co-chaperones, foldases or other components of the proteostasis network because reprogramming readies the folding machinery and up regulates the essential folding components beforehand thus  maintaining system capability of the folding machinery.

The Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli (July 2014)

 

 Unfolded proteins collapse when exposed to heat and crowded environments

Proteins are important molecules in our body and they fulfil a broad range of functions. For instance as enzymes they help to release energy from food and as muscle proteins they assist with motion. As antibodies they are involved in immune defence and as hormone receptors in signal transduction in cells. Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions. Surprisingly, it has been shown that many important proteins occur as unfolded coils. Researchers seek to establish how these disordered proteins are capable at all of assuming highly complex functions.

Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect.

Measurements using the “molecular ruler”

“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.

With computer simulations the researchers have mimicked the behaviour of disordered proteins.
(Courtesy of Jose EDS Roselino, PhD.

 

MLKL compromises plasma membrane integrity

Necroptosis is implicated in many diseases and understanding this process is essential in the search for new therapies. While mixed lineage kinase domain-like (MLKL) protein has been known to be a critical component of necroptosis induction, how MLKL transduces the death signal was not clear. In a recent finding, scientists demonstrated that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death.

They also found a patch of positively charged amino acids on the surface of the 4HBD that bound to phosphatidylinositol phosphates (PIPs) and allowed the recruitment of MLKL to the plasma membrane that resulted in the formation of pores consisting of MLKL proteins, due to which cells absorbed excess water causing them to explode. Detailed knowledge about how MLKL proteins create pores offers possibilities for the development of new therapeutic interventions for tolerating or preventing cell death.

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates (May 2014)

 

Mitochondrial and ER proteins implicated in dementia

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations that facilitate a number of cellular functions. However, the molecular mechanisms of these interactions aren’t properly understood.

A group of researchers showed that the ER protein VAPB interacted with mitochondrial protein PTPIP51 to regulate ER-mitochondria associations and that TDP-43, a protein implicated in dementia, disturbs this interaction to regulate cellular Ca2+ homeostasis. These studies point to a new pathogenic mechanism for TDP-43 and may also provide a potential new target for the development of new treatments for devastating neurological conditions like dementia.

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nature (June 2014)

 

A novel strategy to improve membrane protein expression in Yeast

Membrane proteins play indispensable roles in the physiology of an organism. However, recombinant production of membrane proteins is one of the biggest hurdles facing protein biochemists today. A group of scientists in Belgium showed that,

by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis,

enhanced expression of recombinant membrane proteins in yeast is achieved.

Specifically, they engineered the oleotrophic yeast, Yarrowia lipolytica, by

deleting the phosphatidic acid phosphatase, PAH1 gene,

which led to massive proliferation of endoplasmic reticulum (ER) membranes.

For all 8 tested representatives of different integral membrane protein families, they obtained enhanced protein accumulation.

 

An unconventional method to boost recombinant protein levels

MazF is an mRNA interferase enzyme in E.coli that functions as and degrades cellular mRNA in a targeted fashion, at the “ACA” sequence. This degradation of cellular mRNA causes a precipitous drop in cellular protein synthesis. A group of scientists at the Robert Wood Johnson Medical School in New Jersey, exploited the degeneracy of the genetic code to modify all “ACA” triplets within their gene of interest in a way that the corresponding amino acid (Threonine) remained unchanged. Consequently, induction of MazF toxin caused degradation of E.coli cellular mRNA but the recombinant gene transcription and protein synthesis continued, causing significant accumulation of high quality target protein. This expression system enables unparalleled signal to noise ratios that could dramatically simplify structural and functional studies of difficult-to-purify, biologically important proteins.

 

Tandem fusions and bacterial strain evolution for enhanced functional membrane protein production

Membrane protein production remains a significant challenge in its characterization and structure determination. Despite the fact that there are a variety of host cell types, E.coli remains the popular choice for producing recombinant membrane proteins. A group of scientists in Netherlands devised a robust strategy to increase the probability of functional membrane protein overexpression in E.coli.

By fusing Green Fluorescent Protein (GFP) and the Erythromycin Resistance protein (ErmC) to the C-terminus of a target membrane protein they wer e able to track the folding state of their target protein while using Erythromycin to select for increased expression. By increasing erythromycin concentration in the growth media and testing different membrane targets, they were able to identify four evolved E.coli strains, all of which carried a mutation in the hns gene, whose product is implicated in genome organization and transcriptional silencing. Through their experiments the group showed that partial removal of the transcriptional silencing mechanism was related to production of proteins that were essential for functional overexpression of membrane proteins.

 

The role of an anti-apoptotic factor in recombinant protein production

In a recent study, scientists at the Johns Hopkins University and Frederick National Laboratory for Cancer Research examined an alternative method of utilizing the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, specifically, through the co-transfection of Bcl-xL along with the product-coding target gene.

Chinese Hamster Ovary(CHO) cells were co-transfected with the product-coding gene and a vector containing Bcl-xL, using Polyethylenimine (PEI) reagent. They found that the cells co-transfected with Bcl-xL demonstrated reduced apoptosis, increased specific productivity, and an overall increase in product yield.

B-cell lymphoma-extra-large (Bcl-xL) is a mitochondrial transmembrane protein and a member of the Bcl-2 family of proteins which are known to act as either pro- or anti-apoptotic proteins. Bcl-xL itself acts as an anti-apoptotic molecule by preventing the release of mitochondrial contents such as cytochrome c, which would lead to caspase activation. Higher levels of Bcl-xL push a cell toward survival mode by making the membranes pores less permeable and leaky.

 

 

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What is the meaning of so many RNAs?

Writer and Curator: Larry H. Bernstein, MD, FCAP

 

The following is a third in the 2nd series that is focused on the topic of the impact of genomics and transcriptomics in the evolution of 21st century of medicine, We have already visited the transcription process, by which an RNA sequence is read.  This is essential for protein synthesis through the ordering of the amino acids in the primary structure. However, there are microRNAs and noncoding RNAs, and there are transcription factors.  The transcription factors bind to chromatin, and the RNAs also have some role in regulating the transcription process. We shall examine this further.

  • RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

  • The role and importance of transcription factors?

Larry H. Bernstein, MD, FCAP, Writer and Curator https://pharmaceuticalintelligence.com/2014/8/05/What-is-the-meaning-of-so-many-RNAs

  • What is the meaning of so many RNAs?

Larry H. Bernstein, MD, FCAP, Writer and Curator https://pharmaceuticalintelligence.com/2014/8/05/What-is-the-meaning-of-so-many-RNAs

  • Pathology Emergence in the 21st Century

Larry Bernstein, MD, FCAP, Author and Curator https://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/

  • The Arnold Relman Challenge: US HealthCare Costs vs US HealthCare Outcomes

Larry H. Bernstein, MD, FCAP, Reviewer and Curator; and Aviva Lev-Ari, PhD, RN, Curator https://pharmaceuticalintelligence.com/2014/08/05/the-relman-challenge/

Exploring the Roles of Enhancer RNAs Scientists have recently discovered that enhancers are often transcribed into RNAs. But they’re still not sure what, if anything, these eRNAs do.

By Ashley P. Taylor | May 7, 2014

http://www.the-scientist.com/?articles.view/articleNo/39906/title/Exploring-the-Roles-of-Enhancer-RNAs/

Four mechanisms by which eRNAs can function

Four mechanisms by which eRNAs can function

Four mechanisms by which eRNAs can function Wikimedia, PClermont There’s a lot that scientists don’t yet know about enhancers, genetic elements first described almost 35 years ago that, unlike promoters,

  • can upregulate genes from some distance.

That distance, while generally under 100 kilobases, can vary greatly. Usually,

  • enhancers regulate the genes closest to them,

but not always; the enhancer for the developmental gene Sonic hedgehog is a megabase away from its promoter in the human genome. What scientists do know is that enhancers seem to play key roles in human biology. One recently published atlas of enhancer expression in the human genome suggested that

  • enhancers, which are expressed differently across cell types,

could help explain how one genome encodes so many different kinds of cells. The same paper reported that

  • single-nucleotide changes associated with human diseases are
  • over-represented in enhancers and promoters relative to exons.

In a 2010 Nature paper, researchers in the lab of neurobiologist Michael Greenberg at Harvard Medical School reported that enhancers can produce RNA. Working with cultured mouse neurons, the scientists found that

  • enhancers activated by neuron depolarization were transcribed all over the genome
  • and that levels of enhancer RNAs (eRNAs)
  • correlated with the production of messenger RNA (mRNA)
  • from genes near the enhancers.

Researchers had observed enhancer RNAs before, but this was the first evidence of widespread enhancer transcription. In the years since, several other groups have reported finding eRNAs in various biological systems. While eRNAs promise to help researchers understand how enhancers work, they also raise many questions of their own. ERNAs are fairly short, ranging in length from 500 basepairs to 5 kilobases. Most of the time, although not always,

  • enhancer RNAs are transcribed from both DNA strands, producing what are called bidirectional transcripts.

As the Greenberg lab originally found,

  • eRNA production correlates with the production of mRNA from the genes that enhancers regulate.

“Perhaps the best mark of an active enhancer is the induction of an enhancer RNA,” said M. Geoffrey Rosenfeld from the University of California, San Diego, whose group studies genome-wide regulation of gene expression and has been probing eRNA function. Of course, correlation does not equal causation, he warned. “The next question is whether the enhancer RNA is just a mark of an active enhancer or if it could have function, per se.” What might those functions be? Scientists have crafted a few hypotheses, although most agree that the mechanisms by which eRNAs function remain a mystery. One thought is that eRNAs represent transcriptional noise. That’s a view held by Albin Sandelin at the Bioinformatics Centre of Copenhagen University, who led the aforementioned enhancer atlas project. “Something that most people believe is that the enhancer, when it’s active, it loops in to the promoter, and then the concentration of RNA polymerase II will be a lot higher,” Sandelin told The Scientist. He referred to the idea that

  • enhancers upregulate genes by physically interacting with target promoters;
  • as enhancers and the promoters they regulate are usually on the same chromosome,
  • this interaction creates a loop of DNA between the two genetic elements.

“I think that most of the eRNAs are due to this: you have piece of open DNA, which is the enhancer, near a promoter with high concentrations of RNA polymerase II, and

  • then you will get transcription of the enhancer.”

This idea is supported by the fact that, because they are so short, eRNAs tend to degrade rather quickly. “There are a few cases where [enhancer RNAs] are proven to be functional; I just personally don’t think that the majority work that way,” Sandelin said. “[But] it seems to me that this [supposed function] is a byproduct of proximity to some sites that have a lot of polymerase II.” Another hypothesis suggests that

  • the act of transcription trumps the importance of the transcripts themselves.

Experiments in macrophages, led by the University of California, San Diego’s Chris Glass, support this idea. “I don’t think that transcription at enhancers is noise,” said Vittorio Sartorelli, a researcher at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) who has studied eRNAs in the context of myogenesis. “Whether eRNAs are absolutely required in every situation or the act of transcription at enhancers is the major determinant, I think that it’s still open for debate,” he said. Several studies have presented evidence to suggest that eRNAs do play a key role. As described in a 2013 Nature paper, the Rosenfeld lab found that estrogen bound to its receptor in breast cancer cells increases expression of particular genes by binding to enhancers and inducing the transcription of eRNAs. Eliminating the eRNAs using both RNA interference (RNAi) and antisense oligonucleotides—

  • which bind to the RNA and lead to its degradation by an RNase—
  • reduced the ability of enhancers to upregulate gene expression.

Depletion of eRNAs in this system also

  • reduced looping between enhancers and promoters.

“Using two different strategies, there [was] evidence that

  • the presence of the interrogated eRNA is important for activation of the target gene
  • and for the interaction between the enhancer and the cognate regulated promoter,” said Rosenfeld.

Taking the reverse approach, the researchers also tested whether eRNAs could upregulate transcription independent of the enhancer itself. Rosenfeld and his colleagues experimentally

  • tethered eRNAs to a promoter driving expression of a luciferase reporter gene in a plasmid construct. When this construct was
  • transfected into cultured breast cancer cells,
    • luciferase expression was upregulated about 2.5-fold.

“These data indicate that

  • the interrogated eRNA plays a functional role, at least in this system,
  • in activation of the coding target gene,”

said Rosenfeld. “But it does not distinguish between the alternative possibilities that this is because of a specific sequence in the eRNA that might interact with a regulatory factor or that some other function of the eRNA …” Scientists, such as Reuven Agami from the Netherlands Cancer Institute, the NIAMS’s Sartorelli, and San Diego’s Glass, have reported similar results in other systems. Greenberg said that some eRNAs could have functions unrelated to the promoters activated by their associated enhancers while others could play direct roles in gene expression. “I think that we need to keep an open mind as to what the functions are; there are likely to be multiple functions.” Tags: transcriptional       regulationtranscriptionRNAipromotersmRNAEnhancers and DNA   SiRNA-Mediated Down-Regulation of Livin Expression in Breast Cancer Cells Hussein Sabit, Mohamed M.M. Ibrahim and Nabil S. Awad 1College of Biotechnology, Misr University for Science and Technology, Egypt 2Scientific Research Deanship, Taif University, KSA Academic Journal of Cancer Research 6 (2): 69-73, 2013  http://dx.doi.org:/10.5829/idosi.ajcr.2013.6.2.76211   Livin, also called melanoma inhibitor of apoptosis protein (IAP) or kidney IAP, is an anti-apoptotic protein belonging to the IAP family which consists of eight members. The genes of this family render cancer cells insensitive to apoptotic stimulation. The aim of the present study was to investigate and assess the role of siRNA

  • in the regulation of livin gene expression in two breast cancer cell lines (4Ti and MCF-7).

Lipofection was carried out to introduce the livin-specific small interference RNA (siRNA) segment (19 mer) into the cancerous cells and the livin expression was determined using RT-PCR. Trypan blue assay was conducted to assess the integrity of the cell membranes after being transfected. 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay was also implemented to assess the cell viability through the mitochondrial reductase enzymes activity. The obtained results concluded that

  • transfecting the cancerous cells with livin-specific siRNA have
  • led to the down regulation of livin expression.

RNA interference (RNAi) targeting the anti-apoptotic genes such as livin is a promising approach and may help as a future therapeutic tool for breast cancer. Key words: SiRNA Livin Down-regulation Breast cancer     Endogenous RNA interference is driven by copy number Cristina Cruz, Jonathan Houseley* Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom Cell biology | Genomics and evolutionary biology cruz and Houseley. eLife 2014;3:e01581  http://dx.doi.org:/10.7554/elife.01581   A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate

  • RNA interference (RNAi) responses.

Here we have used a synthetic RNAi system to show that

  • gene copy number is a key factor controlling RNAi for transcripts from endogenous loci,

since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci.

  • Selectivity towards transcripts from high-copy DNA

is therefore an emergent property of a minimal RNAi system. The ability of RNAi to

  • selectively degrade transcripts from high-copy loci
  • would allow suppression of newly emerging transposable elements,

but such a surveillance system requires transcription. We show that

  • low-level genome-wide pervasive transcription
  • is sufficient to instigate RNAi, and propose that
  • pervasive transcription is part of a defense mechanism capable of directing a
  • sequence-independent RNAi response against
  • transposable elements amplifying within the genome.

http://dx.doi.org:/10.7554/eLife.01581.001     Over the past decade, our understanding of the complexity of the eukaryotic transcriptome has been revolutionized. Genome-wide sequencing studies in many organisms have revealed that protein-coding mRNAs are augmented by a multitude of non-protein coding RNAs (ncRNAs), many produced from regions of the genome traditionally considered to be transcriptionally silent (Brummelkamp et al., 2002; Bertone et al., 2004; Cheng et al., 2005; David et al., 2006; Birney et al., 2007). Functional data for the vast majority of ncRNAs are currently lacking, with only a few examples characterized in any detail; however, the diversity of mechanisms by which these act suggests that ncRNAs have a rich and varied biology that is largely still to be sampled. Long ncRNAs which overlap protein-coding genes have the potential to modulate the expression of their cognate coding RNA. Early characterized examples in yeast were thought to work by directly disrupting transcription factor or polymerase binding to the promoter of the coding RNA (Martens et al., 2004; Hongay et al., 2006); however, more recent data implicate specific chromatin structure changes in repression (Gelfand et al., 2011; Hainer et al., 2011), and many other cases of ncRNAs that alter chromatin modifications have been described (Camblong et al., 2007; Berretta et al., 2008; Houseley et al., 2008; Pinskaya et al., 2009; van Werven et al., 2012). Chromatin modifications are not necessarily repressive, and ncRNAs that enhance expression of their overlapping coding gene have also been described (Uhler et al., 2007; Hirota et al., 2008).

Frequency of annotated antisense non-protein coding RNAs

Frequency of annotated antisense non-protein coding RNAs

Figure 1. Frequency of annotated antisense non-protein coding RNAs (ncRNAs) and effects on mRNA abundance. (A) Schematic of an example sense mRNA-antisense (ncRNA) system. (B) Number of annotated open reading frames (ORFs) with antisense transcripts. Positions of CUTs, SUTs, and XUTs were collated with expressed ORFs (Xu et al., 2009; van Dijk et al., 2011), SUTs were later re-classified as XUTs were removed. Overlaps between ORFs expressed in glucose media (total 5171, Xu et al., 2009) and other RNAs were calculated and summed for increasing minimum overlaps of 50–500 bp. ORF–ORF overlaps and ORF–ncRNA overlaps were analyzed separately as ORF–ORF overlaps are consistently smaller. Detailed figures are given in Table 1. (C) Abundance of short interfering RNAs (siRNAs) in RNA interference (RNAi)+ strain produced from expressed ORFs with and without an annotated overlapping antisense ncRNA, based on read counts from published high-throughput sequencing data (Drinnenberg et al., 2011). Minimum antisense overlap with ORF was set at 250 bp; only ORFs with >100 reads in the wild-type poly(A)+ library were assessed to remove noise. Stated p value calculated by Student’s t test. (D) Abundance of mRNA in RNAi+ cells relative to wild-type; data source and categories as in C, differences were not significant. http://dx.doi.org:/10.7554/eLife.01581.003

Multi-copy loci are preferentially targeted by RNA interference (RNAi).

Multi-copy loci are preferentially targeted by RNA interference (RNAi).

Figure 4. Multi-copy loci are preferentially targeted by RNA interference (RNAi). (A) Short interfering RNA (siRNA) (Drinnenberg et al., 2011) and total RNA (Silva et al., 2002) abundance for loci with copy number <2 (left, single-copy) or ≥2 (right, multi-copy). (B) Quantification of data from A binned into categories of increasing total RNA level, with p values for pairwise comparisons of siRNA abundance in single-copy and multi-copy datasets using the Wilcoxon Rank Sum test. (C) Copy number distribution of the 1% of loci with the highest siRNA:total RNA ratio compared with other loci; difference is significant by Wilcoxon Rank Sum test, p<2.2 × 10−16, loci scoring below noise threshold (0–2 category in B) were removed. n values for tests in B and C are given in Table 2. (D) Comparison of copy number with siRNA:total RNA ratio across chromosome I. Cruz and Houseley. eLife 2014;3:e01581. http://dx.doi.org:/10.7554/elife.01581   eLife digest Genes contain the codes that are needed to make the proteins used by cells. This code is transcribed to make a messenger RNA molecule that is then translated to make a protein. However, other types of RNA called

  • non-coding RNA molecules can disrupt this process
  • by binding to messenger RNA molecules,
  • with matching sequences, before translation begins.

RNA interference involves enzymes called Dicer and Argonaute. Many cells contain large numbers of non-coding RNA molecules—

  • so called because they are not translated to produce proteins—
  • and many of these are capable of starting the process of RNA interference.

However, most do not, and the reasons for this are not understood. Now, work by Cruz and Houseley has provided new insight into this phenomenon by showing that

  • it is related to the number of copies of the gene encoding such RNAs in the genome.

Yeast cells normally do not have the genes for RNA interference, but Cruz and Houseley used

  • genetically engineered yeast cells containing Dicer and Argonaute.

Although most of the messenger RNA molecules in these cells showed no change,

  • the expression of some genes with high ‘copy numbers’ was reduced.

Further experiments that involved adding more and more copies of other genes showed that

  • RNA interference could selectively target messenger RNA molecules produced from genes with an increased copy number—
  • particularly if the copies of the genes were clustered in one location in the genome.

RNA interference is also used to defend against DNA sequences that invade and multiply within a genome, such as viruses and other ‘genetic parasites’. As such, the effect observed by Cruz and Houseley could explain why entire genomes are often continuously copied to RNA at low levels. This activity would allow the monitoring of the genome for the invasion of any genetic parasites that had multiplied to high numbers. Following on from this work, the next challenge will be to understand how gene copy number and location are balanced to achieve a selective RNA interference system.   RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts Sarah Geisler1,2 and Jeff Coller1 1Center for RNA Molecular Biology, Case Western Reserve, Cleveland , OH 2Present address: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zürich, 4058 Basel, Switzerland. NATURE REVIEWS | MOLECULAR CELL BIOLOGY

  1. nature.com/reviews/molcellbio http://dx.doi.org:/10.1038/nrm3679

Abstract | The increased application of transcriptome-wide profiling approaches has led to an explosion in the number of documented long non-coding RNAs (lncRNAs). While these new and enigmatic players in the complex transcriptional milieu are encoded by a significant proportion of the genome, their functions are mostly unknown. Early discoveries support a paradigm in which   lncRNAs regulate transcription via chromatin modulation, but

  • new functions are steadily emerging.

Given the biochemical versatility of RNA, lncRNAs may be used for various tasks, including

  • post-transcriptional regulation,
  • organization of protein complexes,
  • cell-cell signalling and
  • allosteric regulation of proteins.

Nature Reviews – Molecular Cell Biology | 9 Oct 2013; http://dx.doi.org:/10.1038/nrm3679   In this Review, we focus on the functional attrib­utes of RNA and highlight the unconventional, and perhaps underappreciated, biological contributions of lncRNAs, including the diverse mechanisms through which lncRNAs participate in transcriptional regu­lation. We touch briefly on the roles of lncRNAs in regulating chromatin states, as this has been explored in several recent reviews (see REFS 8,9,13–15). In addi­tion, we highlight roles beyond transcription whereby lncRNAs function in various cellular contexts, includ­ing

  1. post-transcriptional regulation,
  2. post-translational regulation of protein activity,
  3. organization of protein complexes,
  4. cell–cell signalling, as well as
  5. recombination

Transcription activator-like effectors (TALEs). Naturally found in some bacteria, TALEs are proteins that bind DNA through repeat domains, and their code for sequence specificity has been elucidated allowing sequence specific TALEs to be engineered.

  • PUF proteins

A family of sequence-specific RNA-binding proteins, which bind 3ʹ untranslated regions within mRNAs to repress target mRNA translation.

  • Pseudogenes

Dysfunctional relatives of normal genes thought to arise from duplication or retrotransposition.

  • Chromatin-modifying complexes

Protein complexes that catalyse the covalent chemical modification of chromatin

  • Adaptive immune system

A system of specialized cells that create immunological memory via specific antibodies after an initial response to a pathogen.   A biochemically versatile polymer   Figure 1 | RNA is a biochemically versatile polymer. a | RNA is particularly well suited for sequence-specific nucleic acid targeting through base pairing interactions over a short region (for example, eight nucleotides). By contrast, proteins require repeat motifs comprising 35–39 amino acids (105–117 base pairs of genomic sequence) to recognize a single RNA base with specificity. Therefore, to recognize eight nucleotides, 280–312 amino acids (840–936 base pairs of genomic sequence) would be required. Compared to the eight base pairs required for an RNA, protein-based nucleic acid recognition requires substantially more genomic sequence17. b | RNA can fold into complex three-dimensional structures that can specifically bind various ligands, including small molecules and peptides18. c | RNA is suitable for transient expression, because a fully functional RNA can be generated immediately following transcription and processing but can also be rapidly degraded. Together, this allows RNA effectors to be produced in quick pulses. Proteins, however, require additional steps, including mRNA export and translation, to produce a functional peptide. Likewise, both the mRNA and the protein need to be degraded to turn off expression. d | RNA is malleable and, therefore, more tolerant of mutations. Although some mutations in protein-coding genes are silent, many are deleterious such as nonsense mutations that generate truncated polypeptides. RNA, however, can tolerate mutations even within the regions responsible for target recognition. e | RNA-dependent events can be heritable. For instance, processed pseudogenes were once RNA transcripts that have been genomically integrated. In addition, telomerase uses an RNA template to add telomeric repeats to the ends of chromosomes. ORF, open reading frame; Pol II, RNA polymerase II.   lncRNAs as regulators of transcription   Figure 2 | lncRNAs regulate transcription through several mechanisms. ac | Long non-coding RNAs (lncRNAs) can modulate chromatin through transcription-independent (part a) and transcription-dependent mechanisms (parts b and c). lncRNAs can bind one or more chromatin-modifying complexes and target their activities to specific DNA loci (part a). Depending on the nature of the enzymes bound, lncRNA-mediated chromatin modifications can activate or repress gene expression22,23,26,27,120. Chromatin-modifying complexes bound to the RNA polymerase II (Pol II) carboxy-terminal domain (CTD) can modify chromatin during transcription of lncRNAs33–35 (part b). Transcription of lncRNAs can also result in chromatin remodelling that can either favour or inhibit the binding of regulatory factors (part c). Depending on the nature of the factors that bind during remodelling, gene expression is activated or repressed 37–40. dg | lncRNAs can modulate both the general transcription machinery (parts d and e) as well as specific regulatory factors (parts f and g). lncRNAs can bind Pol II directly to inhibit transcription47 (part d). Formation of lncRNA–DNA triplex structures can also inhibit the assembly of the pre-initiation complex48 (part e). lncRNAs can fold into structures that mimic DNA-binding sites (left) or that generally inhibit or enhance the activity of specific transcription factors (right)50–53 (part f). lncRNAs can also regulate gene expression by binding specific transport factors to inhibit the nuclear localization of specific transcription factors54 (part g). Regulators of mRNA processing Modulators of post-transcriptional control   Figure 3 | lncRNAs influence mRNA processing and post-transcriptional regulation. a,b | Long non-coding RNAs (lncRNAs) can modulate mRNA processing. Splicing patterns can be influenced by lncRNAs that associate with the pre-mRNA (part a). For example, splicing of the first intron of neuroblastoma MYC mRNA is prevented by a natural antisense transcript61. Antisense lncRNAs that associate with an mRNA could direct mRNA editing, perhaps through association of the duplex with ADAR (adenosine deaminase acting on RNA) enzymes that catalyse adenosine to inosine conversion in double-stranded RNA63,66 (part b). cf | lncRNAs modulate post-transcriptional regulatory events. lncRNAs containing SINEB2 repeat elements can upregulate translation through association with the 5ʹ region of an mRNA68 (part c). lncRNAs containing Alu repeat elements associate with the Alu elements in the 3ʹ untranslated region (UTR) of an mRNA, and this double-stranded structure can direct Staufen-mediated decay through a pathway that is molecularly similar to nonsense-mediated decay70 (part d). lncRNAs can mask miRNA-binding sites on a target mRNA to block miRNA-induced silencing through the RNA-induced silencing complex (RISC)72 (part e). Linear or circular lncRNAs can function as miRNA decoys to sequester miRNAs from their target mRNAs74,75 (part f).                   Regulators of protein activity                 Scaffolds for higher-order complexes                 Signaling molecules   Figure 4 | lncRNAs are involved in various cellular contexts. Long non-coding RNAs (lncRNAs) modulate protein activity by post-translational mechanisms (parts ac). a | Small nucleolar lncRNAs (sno-lncRNAs) generated from the 15q11‑q13 locus bind and modulate the activity of the FOX2 alternative splicing factor, and this can inhibit FOX2‑mediated splicing80. b | The highly structured rncs‑1 lncRNA binds Dicer to inhibit the processing of small RNAs81. c | The gadd7 lncRNA binds and modulates the ability of TDP43 (TAR DNA-binding protein 43) to target and process specific mRNAs84. d | lncRNAs can act as scaffolds to organize several complexes24. e | As the cargo of exosomes that mediate transfer of material between cells, exosomal shuttle RNAs (exRNAs) may act as signalling molecules during cell–cell communication; exosomal cargo includes mRNAs, microRNAs (miRNAs) and lncRNAs102. f | lncRNAs expressed from the switch region of genes encoding antibodies form R‑loops to direct class switch recombination via activation-induced deaminase (AID) recruitment111                   Vehicles for increasing genetic diversity                 Conclusions and perspectives                 lncRNAs have now been demonstrated to regulate all aspects of gene expression, including transcription (FIG. 2), processing and post-transcriptional control path­ways (FIG. 3). Likewise, lncRNAs have also been shown to regulate protein function and organize multiprotein com­plex assembly. Now with hints that lncRNAs might par­ticipate in cell–cell communication and recombination, the possible reach of lncRNA functions seems endless (FIG. 4).   Targeting Noncoding RNAs in Disease: Challenges and Opportunities Science/AAAS technology webinar   4 Sept 2013  

  • Noncoding RNAs serve a wide range of functions in cellular and developmental processes and are therefore likely involved in the development and pathophysiology of many diseases.

Thanks to the effective inhibition of micro RNAs in vivo, scientists have already made groundbreaking discoveries about the contribution of short regulating RNAs in human diseases in areas such as cancer, heart disease, and diabetes. Dr. David Corey from the Department of Pharmacology at the University of Texas Southwestern Medical Campus in Dallas, Texas; Dr. Stefanie Dimmeler from the Institute of Cardiovascular Regeneration at Goethe‐University in Frankfurt, Germany; Dr. Jan‐Wilhelm Kornfeld from the Department of Mouse Genetics and Metabolism, University of Cologne in Germany.   Dr. Corey’s group is interested in antigene oligonucleotides, antisense oligonucleotides, nucleic acids, RNAi, and telomerase.   I have two goals for my presentation today.

  • give a brief introduction to the concept of using nucleic acids as dugs.
  • show the boundaries of using nucleic acids to affect gene expression

 

  • a search for new methods to develop drugs includes nucleic acids that can bind to RNA and affect gene expression. The advantages of this approach are that one can identify an active oligomer, a lead compound very quickly in weeks rather than years.
  • the medicinal chemistry and pharmacology of all of these nucleic acids is similar
  • by affecting gene expression, one has the ability to treat almost any disease

the two main strategies for using nucleic acid to affect gene expression are

  • use single cell stranded oligonucleotides to bind directly to an RNA target and block their action.
  • use double stranded RNA. Double stranded RNA then goes through the RNA silencing process. That machinery
  • helps it to find a messenger RNA target and efficiently inhibit gene expression.

What kind of cellular RNAs can be targeted by nucleic acids?

  • they could be the RNA domains of ribonucleoproteins and the classic example of that is telomerase.
  • One could also target messenger RNA.
  • You could block translation or you could affect splicing so for example upregulate an isoform that might be useful in treating a disease.
  • Today, we focus mainly on targetingnoncodingRNAs and one of thosenoncodingRNAs ismicroRNAs and by
    • blocking the microRNA you can affect its action.

I’m going to discuss targeting long noncoding RNAs, which can be used to either up or down regulate gene transcription. Earlier this year Kynamro, an antisense oligonucleotide that targets Ap‐B1 messenger RNA, was approved by the food and drug administration. This is a systemically administered oligonucleotide that’s been shown to reduce LDL cholesterol. So it’s the strongest proof to date that synthetic oligonucleotides can be made on the scale that’s large enough to be used as drugs and be administered to patients and get through the FDA approval process.

  • Now I’d like to show you just how the boundaries of regulation can be pushed by using noncoding RNAs to regulate transcription of an operon within the icosanoid signaling pathway. Messenger RNAs are often overlapped by long RNAs at both their 3’ and the 5’ termini as well as within the gene providing a new realm of potential targets for addressing gene expression.

 

  • examples of the important ones include XIST and HOTAIR
  • These are genes that are known to regulate x chromosome inactivation or transcriptional multigene regulation programs. one might think that with RNA that RNAi factors that are so successful in regulating messenger RNA might be involved. But today they haven’t really been strongly implicated in mammalian cells.

 

  •  we know that microRNAs are in the nucleus.
  • we also know that RNAi factors like argonaute 2 are in the nucleus.
  • we also know that noncoding RNAs are in the nucleus.

The hypothesis that we’ve built up over that time is that these RNAi factors can interact with small RNAs

  • to form what are essentially ribonucleoprotein complexes that can act to control either gene transcription or gene splicing.
  • The RNA domain protects the RNA and promotes binding to the target. The RNA domain directs specificity to a particular RNA target inside the cell, for example a long noncoding RNA.

in about 2010 working with my colleague Bethany Janowski, we decided to go very deeply into an important physiology pathway — the eicosanoid production pathway and cyclooxygenase‐2 and PLA2G4a

  • we began by asking whether or not there was noncoding expression at the COX‐2 promoter
  • we characterized this expression by RNA sequencing by quantitative PCI and 5’ RACE and
  • we discovered that there were transcripts overlapping the COX-promoter in both the antisense and the sense direction –
  • we now have the noncoding RNA raw material that might allow recognition to control gene expression of COX‐2 messenger RNA.

 

  • there were a substantial number of microRNAs with complementarity to the COX‐2 promoter.
  • small RNA sequencing to identify microRNAs in the nucleus and
  • microRNAs that were both in the nucleus and complementary to the

COX‐2 promoter became candidates for regulating COX‐2.

  • The most promising of these microRNAs was microRNA‐
  • It had strong complementarity to two adjacent sequences within the COX‐2

promoter. So that resembles how micro‐RNAs recognize typically 3’ untranslated regions.

  • it became our prime candidate for investigating for potential regulation of COX‐2 expression through regulating its transcription by binding a noncoding RNA.
  •  we used a microRNA inhibitor
  • When we add these inhibitors into cells COX‐2 expression goes down.
  • consistent with a microRNA binding to the noncoding RNA and activating COX‐2 expression

This is as far as this reviewer wishes to prodeed in the presentation(s)   Explore microRNA as therapeutic targets Efficient [in vivo] silencing using LNA™-enhanced inhibitors exiqon.com/in-vivo-mirna-inhibitors   Nature Reprint Collection MicroRNAs from bench to clinic   Progress in the microRNA fi eld over the last 12 years has been nothing but remarkable. MicroRNAs were only discovered in humans in 2001, but since then they have revolutionized cell biology and completely changed the way we view the regulation of gene expression. They are now known to be involved, at some level, in all cellular and developmental pathways and all major types of disease, including all cancers, as well as metabolic, cardiovascular, neuronal and immune-related disorders. Exiqon’s LNA™- based microRNA research tools have been instrumental in many of the groundbreaking discoveries in the field. In this collection, we are thrilled to present some of the recent advances in moving microRNAs from basic research into the clinic both as biomarkers and therapeutic targets. Since the discovery of circulating or extracellular microRNAs, their potential as minimally invasive diagnostic and prognostic markers for disease has been actively investigated. Here we feature two articles where qPCR profiling of microRNAs in biofluids have been shown to have diagnostic potential. Another promising area with clinical prospects is microRNA in situ hybridization (ISH) in FFPE samples. We have included an article detailing the prognostic potential of microRNA ISH in this collection. Due to their extensive involvement in human disease, microRNAs are naturally interesting targets for therapeutic intervention. One of the most advanced areas in this respect is the potential of microRNAs as therapeutic targets in cardiovascular disease and we have included a review of this area. In addition, two very recent and groundbreaking studies that have shown the exciting potential for microRNA inhibition in diabetes and epilepsy are also included.   Identification of serum microRNA profiles in colon cancer E Hofsli*,1,2,7, W Sjursen3,4,7, W S Prestvik5, J Johansen2, M Rye2, G Tranø6, et al. 1Department of Oncology, St Olavs Hospital, Trondheim University Hospital, 2Faculty of Medicine, Department of Cancer and Molecular Medicine, Norwegian University of Science and Technology,  3Department of Laboratory Medicine Children’s and Women’s Health, Norwegian University of Science and Technology, 4Department of Pathology and Medical Genetics, St Olavs Hospital, Trondheim University Hospital, 5 Faculty of Technology, Sør-Trøndelag University College, and 6Department of Gastrointestinal Surgery, St Olavs Hospital, Trondheim University Hospital, Olav Kyrresgt 17, Trondheim 7006, Norway British Journal of Cancer (2013) 108, 1712–1719 |  http://dx.doi.org/10.1038/bjc.2013.121 Background: microRNAs (miRNAs) exist in blood in an apparently stable form. We have explored whether serum miRNAs can be used as non-invasive early biomarkers of colon cancer. Methods: Serum samples from 30 patients with colon cancer stage IV and 10 healthy controls were examined for the expression of 375 cancer-relevant miRNAs. Based on the miRNA profile in this study, 34 selected miRNAs were measured in serum from 40 patients with stage I–II colon cancer and from 10 additional controls. Results: Twenty miRNAs were differentially expressed in serum from stage IV patients compared with controls (Po0.01). Unsupervised clustering revealed four subgroups; one corresponding mostly to the control group and the three others to the patient groups. Of the 34 miRNAs measured in the follow-up study of stage I–II patients, 21 showed concordant expression between stage IV and stage I–II patient. Based on the profiles of these 21 miRNAs, a supervised linear regression analysis (Partial Least Squares Regression) was performed. Using this model we correctly assigned stage I–II colon cancer patients based on miRNA profiles of stage IV patients. Conclusion: Serum miRNA expression profiling may be utilised in early detection of colon cancer. MicroRNAs from bench to clinic   Figure 2. Differentially expressed miRNAs in stage IV (red bars) vs stage I–II (blue bars) colon cancer. The expression of 34 miRNAs was compared, and 26 miRNAs were detected. In all, 21 of 26 detected miRNAs showed the same expression profile in early-stage I–II vs metastatic stage IV colon cancer.     Figure 3. Prediction analysis of early-stage colon cancer patients. Controls are shown in red and cancer samples in blue. 9 out of 10 healthy controls were correctly predicted as true negatives and 35 out of 40 patients with cancer as true positives.     MicroRNA profiling of diagnostic needle aspirates from patients with pancreatic cancer S Ali1, H Saleh2,3, S Sethi2, FH Sarkar1,2 and PA Philip*,1 1Department of Oncology; 2Department of Pathology; 3Karmanos Cancer Institute, Detroit Medical Center, Wayne State University School of Medicine, Detroit, MI   BACKGROUND: A major challenge to the development of biomarkers for pancreatic cancer (PC) is the small amount of tissue obtained at the time of diagnosis. Single-gene analyses may not reliably predict biology of PC because of its complex molecular makeup.MicroRNA (miRNA) profiling may provide a more informative molecular interrogation of tumours. The primary objective of this study was to determine the feasibility of performing miRNA arrays and quantitative real-time PCR (qRT– PCR) from archival formalin fixed paraffin-embedded (FFPE) cell blocks obtained from fine-needle aspirates (FNAs) that is the commonest diagnostic procedure for suspected PC. METHODS: MicroRNA expression profiling was performed on FFPE from FNA of suspicious pancreatic masses. Subjects included those who had a pathological diagnosis of pancreatic adenocarcinoma and others with a non-malignant pancreatic histology. Exiqon assay was used to quantify miRNA levels and qRT–PCR was used to validate abnormal expression of selected miRNAs. RESULTS: A total of 29 and 15 subjects had pancreatic adenocarcinoma and no evidence of cancer, respectively. The RNA yields per patient varied from 25 to 100 ng. Profiling demonstrated deregulation of over 228 miRNAs in pancreatic adenocarcinoma of which the top 7 were further validated by qRT–PCR. The expression of let-7c, let-7 f, and miR-200c were significantly reduced in most patients whereas the expression of miR-486-5p and miR-451 were significantly elevated in all pancreas cancer patients. MicroRNAs let-7d and miR-423-5p was either downregulated or upregulated with a significant inter-individual variation in their expression. CONCLUSION: This study demonstrated the feasibility of using archival FFPE cell blocks from FNAs to establish RNA-based molecular signatures unique to pancreatic adenocarcinoma with potential applications in clinical trials for risk stratification, patient selection, and target validation. British Journal of Cancer (2012) 107, 1354–1360.  http://dx.doi,org:/10.1038/bjc.2012.383 Comparative expression of seven miRNAs tested in FNA samples   Figure 1 Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs analysed by miRNA profiling in PC and their targeted genes.

miRNAs analysed by miRNA profiling in PC and their targeted genes

miRNAs analysed by miRNA profiling in PC and their targeted genes

Figure 2 Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes (A). The solid lines connecting genes represent a direct relation and dotted lines indirect relation. We also observed 15 bio functional network groups that included cancer, genetic disorder, and gastrointestinal disease (B).

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

Figure 6 Box plot representing the expression of 7 miRNAs as assessed by qRT–PCR in 29 FNA cell blocks from PC patients analysed individually compared with FNA cell blocks obtained from 15 normal controls by using qRT–PCR. The graph is presented in log2 values and 1.0 represents average of normal subjects (n¼15). [not shown]   The prognostic importance of miR-21 in stage II colon cancer:a population-based study S Kjaer-Frifeldt*,1,2, TF Hansen1, BS Nielsen3, S Joergensen3, J Lindebjerg4, …on behalf of Danish Colorectal Cancer Group 1Department of Oncology, Danish Colorectal Cancer Group South, Vejle Hospital; 2University of Southern Denmark, Odense, Denmark; 3Diagnostic Product Development, Exiqon A/S, Vedbæk 2950, Denmark; 4Department of Clinical Pathology, Vejle Hospital, Vejle, Denmark British Journal of Cancer (2012) 107, 1169–1174   BACKGROUND: Despite several years of research and attempts to develop prognostic models a considerable fraction of stage II colon cancer patients will experience relapse within few years from their operation. The aim of the present study was to investigate the prognostic importance of miRNA-21 (miR-21), quantified by in situ hybridisation, in a unique, large population-based cohort. PATIENTS AND METHODS: The study included 764 patients diagnosed with stage II colon cancer in Denmark in the year 2003. One section from a representative paraffin-embedded tumour tissue specimen from each patient was processed for analysis of miR-21 and quantitatively assessed by image analysis. RESULTS: The miR-21 signal was predominantly observed in fibroblast-like cells located in the stromal compartment of the tumours. We found that patients expressing high levels of miR-21 had significantly inferior recurrence-free cancer-specific survival (RF-CSS): HR¼1.26; 95% CI: 1.15–1.60; Po0.001. In Cox regression analysis, a high level of miR-21 retained its prognostic importance and was found to be significantly related to poor RF-CSS: HR¼1.41; 95% CI: 1.19–1.67; Po0.001. CONCLUSION: The present study showed that increasing miR-21 expression levels were significantly correlated to decreasing RF-CSS. Further investigations of the clinical importance of miR-21 in the selection of high-risk stage II colon cancer patients are merited. British Journal of Cancer (2012) 107, 1169–1174. http://dx.doi.org:/10.1038/bjc.2012.365

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