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Author: Margaret Baker, PhD, Registered Patent Agent

The Encyclopedia of DNA Elements (ENCODE) Project was launched in September of 2003. In 2007 the ENCODE project was expanded to study the entire human genome, Genome-wide association studies or GWAS, and published a Nature paper entitled “An integrated encyclopedia of DNA elements in the human genome,” this month also all data are available at http://genome.ucsc.edu/ENCODE/.  Novel functional roles have been discovered for both transcribed and non-transcribed portions of DNA.  See several articles and commentary in Science 7 September 2012: Vol. 337 no. 6099 including Maurano et al. pp. 1190-1195  DOI: 10.1126/science.1222794b

For the first time, the 3-dimensional connections that cross the genome have been mapped as long-range looping interactions between functional elements and the genes controlled. These regions of the genome, formerly referred to as “junk DNA”, have the potential to be involved in disease initiation, pathophysiology, and complications. Further, epigenetic factors may be seen to play a more direct role in the expression or silencing of protein coding genes as DNase I hot spots, nucleosomal anchor points, and DNA methylation sites are added to the map.

Non-coding transcribed DNA includes a large percentage of sequences coding for RNA. In fact, RNA encoding genes number nearly equal to the protein encoding genes- 18,400 v 20,687 – and previously unknown non-coding RNA (ncRNA) have also been characterized.

Some of the known elements that were cataloged include:

• cis elements – promoters, transcription factor binding sites;
• gene contiguous non-coding stretches such as introns, polyA, and UTR, splice variants;
• pseudogenes (11,224);
• long range gene associated elements – enhancers, insulators, suppressors, and predicted promoter flanking regions;
• ribosomal RNA genes; and
• sequences for 7,052 small RNAs of which 85% are small nuclear(sn)RNA, small nucleolar(sno)RNA), transfer(t)RNA, and micro(mi)RNA.

What has been found is that distinct non-coding regions, including ncRNA, can be associated with distinct disease traits. miRNA are among the non-gene encoding sequences in the genome which have already been shown to play a major post-transcriptional role in expression of multiple genes..

Most miRNA genes are intergenic or oriented antisense to neighboring genes and therefore assumed to be controlled by independent promoter units. However, in some cases a microRNA gene is transcribed together with its target gene implying coupled regulation of miRNA and protein-coding gene. About one third of miRNA genes reside in polycistronic clusters. miRNA genes can occupy the introns of protein, non-protein coding genes, or nonprotein-coding transcripts. The promoters have been shown to have some similarities in their motifs to promoters of other genes transcribed by RNA polymerase II such as protein coding genes. The ENCODE project also noted that miRNA promoters were in chromatin regions of high promiscuity. There may be up to 1000 miRNA genes in the human genome. In addition, human miRNAs show RNA editing of sequences to yield products different from those encoded by their DNA.  miRNA are implicated in cellular roles as diverse as developmental timing in worms, cell death and fat metabolism in flies, haematopoiesis in mammals, and leaf development and floral patterning in plants

The final miRNA gene product is a ∼22 nt functional RNA molecule. The mature miRNA (designated miR-#) is processed from a characteristic stem–loop sequence (called a pre-mir), which in turn may be excised from a longer primary transcript (or pri-mir). It is processed by the same enzyme (DICER) that processes short hairpin RNA, forming interfering RNA, which provides and additional level of control.

MiRNA controls gene expression by binding to complementary regions of messenger transcripts in the 3’ untranslated region to repress their translation or regulate degradation. What makes the mechanism more powerful (or complicated) is the imperfect but specific binding motif associates with a large number of mRNAs in the 3’ untranslated region having the complimentary motif.  Conversely then, each mRNA can potentially associate with a number miRNA. Mature processed cytosolic miRNA can act in a manner akin to small interfering(si)RNA, and form the RNA-induced silencing complex (RISC) to block translation. Computational methods have been used to identify potential gene targets based on complimentarity between the miRNA and mRNA sequences.

Gerstein et al. explored the “Architecture of the human regulatory network derived from ENCODE data” Nature 489:91-100 (06 Sep 2012) focusing on the regulation of transcription factors (TF) and association between TF and miRNAs, miRNA and miRNA, protein-protein interactions, and protein phosphorylation. Not surprisingly, not all TF are the upstream factor in each network.

These new and remarkably detailed examinations of the different elements within and transcribed from the human genome perhaps do more to aid our knowledge of why we have stumbled in attempts to eradicate diseases, initially by focusing on a single gene or constellation of coding regions. The miRNA wikipedia is also being re-written on a daily basis and new disease associations made*.  As an example of a pathological state that may be linked to miRNA controlled elements, in vitro as well as in small population studies have examined miRNA species in diabetogenic conditions and patients with diabetes (Type I and Type II).

Diabetes and miRNA

In adult β-cell islets, miR-375 is low when glucose is freely available and low miR-375 induces insulin secretion. Interestingly, miR-375 is found only in brain and β-cells which share a secretion pathway.

Diabetic Complications

Organ specific miRNA have been identified in liver, skeletal muscle, kidney, vascular, and adipose tissue which are responsive to transient or sustained hyperglycemia.

miR-17-5p and miR-132 were reported to show significant differences between obese and non obese omental fat and were also abnormal in the blood of obese subjects.  Altered expression of miR-17-5p and miR-132 were found to correlate significantly with BMI, fasting blood glucose and glycosylated hemoglobin. (Kloting et al. PLoS ONE 4(3), e4699 (2009).

Clinical practice related to miRNA in diabetes may be possible as one group has identified eight miRNAs (miR-144, miR-146a, miR-150, miR-182, miR-192, miR-29a, miR-30d and miR-320) as potential ‘signature miRNAs’ that could distinguish prediabetic patients from those with overt T2D (Karolina DS, Armugam A, Tavintharan S et al. MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in Type 2 diabetes mellitus. PLoS ONE 6(8), e22839 (2011).

Due to the autoimmune component of T1D, the constellation of miRNA would be expected to be different: upregulation of miR-510 and underexpression of miR-191 and miR-342 were observed in the Tregs (regulatory T-cells) of T1D patients (Hezova R, Slaby O, Faltejskova P et al. microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of Type 1 diabetic patients. Cell. Immunol. 260(2),70–74 (2010).

Taken together with the “physical” mapping of miRNA genes in the context of the 3-dimensional genome provided by the ENCODE studies and new understanding of potential concerted regulatory mechanisms, the miRNA data for tissues and specific cell types involved in disease pathology form a new approach to either detecting or possibly correcting gene (coding or non-coding) dysregulation.  miRNA mimics and anti-miRNA agents are being developed as new therapeutic modalities.

References

Bartel, DP et al. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function” Cell 2004, 116:281-297.

Fernandez-Valverde, SL et al. MicroRNAs in beta-cell Biology, insulin resistance, diabetes and its complications. Diabetes July 2011 60 (7):1825-31.

Kantharidis, et al.  Diabetes Complications: The MicroRNA Perspective http://diabetes.diabetesjournals.org/content/60/7/1832.short

MEDSCAPE Review article: “miRNAs and Diabetes Mellitus: miRNAs in Diabetic Complicatons”  http://www.medscape.org/viewarticle/763729_6

*Based on initial studies in the worm C. elegans showing the temporal appearance of 21- and 22-nt RNAs during development, a family of highly conserved micro RNA sequences (miRNA) existing in invertebrates and vertebrates, were cataloged by Tuschl et al. at the Max-Planck-Institute and others (see Eddy, SR  Non-coding RNA genes and the modern RNA world Nature Reviews Genetics, 2:920-929, 2001). The sequence-specific post-transcriptional regulatory mechanisms mediated by these miRNAs have been associated with certain disease states such as cancer miR-21) and more specifically, lung cancer (miR-124) or breast cancer (miR-7, miR-21) and new species and function continue to be found (see http://www.mirbase.org/ ).