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miRNA Science Overview: Their role in biological function & cellular regulation

Introduction

MicroRNA (miRNA) are stable, non-coding, small RNAs about 22 nucleotides in length. They are originally derivative products of genes generated by a eukaryotic RNaseIII nuclease enzyme known as Dicer [6]. The first miRNAs discovered were lin-4 and let-7, identified in Caenorhabditis elegans [17], but it was not until recently that this class of RNA has been extensively studied. Despite the high degree of evolutionary conservation across species revealed by gene clustering, they were previously thought to be molecules lacking an important biological function. This view has changed since miRNA was discovered to be an important regulatory component in eukaryotic gene expression. More significantly, its direct involvement in cancer and viral replication has potential practical implications for medical and pharmaceutical therapeutics.

miRNA-Biogenesis Principle

Figure 1: miRNA Biogenesis and posttranslational silencing mechanism. 'Hannon,G.J et al.' [8].

miRNA was found to be involved in a wide variety of functional regulation in both animal and plant cells via degradation of messenger RNA (mRNA) leading to translational repression. The mechanism by which miRNA silences the target mRNA is similar to that of siRNA (See miRNA versus siRNA). The process is named RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants [7]. The silencing process begins with the activation of an endonuclease-containing complex, named the RNA-induced silencing complex (RISC), by miRNAs. The complex then recognizes the target mRNA, and depending on the formation of perfect or imperfect complementary base pairing, cleaves or represses cognate mRNAs respectively (Figure 1). Finally, the repressed target mRNAs and miRNAs accumulate in cytoplasmic foci (P-bodies) where mRNA will be decayed. It has been suggested that P-bodies sites may serve as repositories of untranslated mRNA [10].

Understanding the dynamic process of miRNA-gene silencing mechanisms based on current research

Current research shows that miRNAs inhibit the function of the cap-binding initiation factor, eIF4E, via base pairing with the 3' UTR (untranslated region) of mRNAs, which eventually results in the attenuation and later termination of the translation stage [10,14]. It is though that the choice between the two pathways (degradation or repression) is determined by the degree of complementarity between miRNAs and their target mRNA. Interestingly, in a recent study by Finnegan et al. it was shown that mRNA degradation by miRNA usually occurs in plants while translational repression occurs in animals. This has been suggested to be due to the formation of perfect complementarity between plant miRNA and their target mRNA, whereas in animals, formation of perfect complementarity with cognate mRNA would be evitable [7]. This model has been further corroborated in an investigation by Babak et al. who used a microarray analysis supporting the hypothesized model that most mammalian miRNAs inhibit the translation process without degradation of the target mRNA [2]. In yet another study, it has been demonstrated that miRNA is involved in regulation of target mRNA despite mismatches and bulges between the two [11].

miRNA versus siRNA

Primitive small RNAs probably originated from transposable elements present in ancestral genomes and further evolved through a series of duplication events followed by dispersal and diversification, akin to the history of protein gene families [19] Unsurprisingly, miRNAs and siRNAs have similar cellular biogenesis and molecular mechanisms for biological functioning, differing only in that miRNA precursors are endogenously encoded by their own genes, while siRNAs are processed from long doublestranded RNA precursors residing exogenously on mRNAs, viruses, or complementary DNA (cDNA) [6].

Applications and uses

Regulation of important cell processes

MiRNAs were found to be involved in regulation of a number of important cellular processes in both animal and plant cells. In invertebrates, they regulate developmental timing, neuronal differentiation (Trk Receptors), cell proliferation, growth control and apoptosis; in mammals, they regulate hematopoietic cell differentiation and brain development [8]; in plants, they regulate developmental patterning such as flowering time as well as stress responses to extreme temperatures [6]. miRNAs were particularly important in cell differentiation. For instance, Kim et al. showed that miR-206 change the gene expression profile of C2C12 toward the differentiation state in muscle cells [11].

Defense against viruses

MiRNA-silencing was hypothesized to present a natural defense mechanism against replication of viruses as miRNA destroys foreign mRNAs [6]. Emerging evidence proved this hypothesis as virus genome was later discovered to encode suppressors that could target the host RNA-silencing mechanism. The first viral miRNA was discovered in Epstein-Barr virus (EBV) in 2004. Soon after, HCMV was found to encode a miRNA that was shown to target a host immune system gene [4].


Cancer diagnosis and biotherapy

Cancer is a consequence of disordered gene expression. Throughout the years many evidence have been suggested that either up-regulation or down-regulation of miRNA could be a leading factor to tumor formation. For instance, let-7 that is an endogenous human miRNA was shown to be a potential tumor suppressor. It was however discovered that up-regulation of let-7 inhibits the expression of RAS protein in human cancer cell lines, which contributed to tumorgenesis [23]. The most recent studies also revealed that down-regulation of several miRNAs contributed to colon carcinomas. As an example, human breast cancer could be a result of dysregulation of an important tumor suppressor protein, Programmed Cell Death 4 (PCD4) by down-regulation of miR-21 [7,9]. Consequently, a vast amount of research has been done to investigate miRNA profiling as a tool for cancer diagnosis and antisense RNA therapies as a cure, if not, a major treatment for cancer. Cancer therapeutics, on the other hand, have major limitations. Oligonucleotides developed to restore functional miRNAs and reduce disease-causing miRNAs, can only be delivered to a restricted number of target cells. Further investigation needs to be done to understand how to chemically modify these antisense RNA in order to allow stability in serum and efficient cellular uptake in vivo [23].

Past and future improvements

The relevance of miRNAs in cancer was underestimated in the past, and the mechanisms by which miRNAs function are still poorly understood. It was doubted whether miRNAs operate more commonly through single, critical target transcripts, or regulate broad programs of unique gene expression through minor effects on multiple targets [23]. The mechanism by which miRNA switch between the two modes of silencing and how small RNAs are channeled into the various pathways by association with the appropriate protein partners remains controversial [7]. In order to understand these critical processes, a miRNA regulatory network needs to be established using Bioinformatics and knockdown analysis [20]. New tools such as microarrays also helped us develop better understanding of the relationship between miRNA and cancer. For instance, an expression profile for cancer diagnosis consisting differentially expressed normal and tumor samples allow us to compare the expression of many miRNAs at once [9]. However, up to now, our knowledge of miRNA has been minimal. More than 200 different mammalian miRNAs have been identified but only a small fraction has assigned target mRNAs or an established role [11]. The mechanism by which miRNA functions and its connection to disease therefore needs to be further defined and will be the future topic of investigation.

Discovery of 'miRNA Timeline'*

Timeline Discovery
1960s Britten and Davidson were first scientists who encountered small RNAs.They proposed a theory in which small RNAs are able to activate protein-coding genes [3]*.
1993 Ambros discovered the first microRNA, lin-4, isolated from C. elegans.[17].
2000 Ruvkun discovered let-7 microRNA, isolated from C. elegans.[21].
2001 Bartel,Tuschl & Ambros Lab collaboration led to understanding of small regulatory, non-coding RNAs. They later named this class of RNA as microRNA [15,13,16].
2001 Later this year Bartel discovered miRNA in plants [22].
2002 Croce showed the interplay of miRNA in many human cancers [5].
2003 Stoffel conducted the first miRNA silencing-induced in vitro [13].


Glossary

  • Antisense: an RNA (antisense) that forms complementary base-pair to mRNA(sense) sequences.
  • Attenuation: A process by which reduces the strength of expression of a target gene.
  • Cytoplasmic foci: Granules found in the cytoplasm of human cells, which serve as sites of mRNA decay.
  • elF: Eukaryotic Initiation Factor.
  • Endonuclease: Restriction enzymes in which recognise bases within the double strand DNA and subsequently cleave at that site.
  • Oligonucleotide: Short nucleic acid polymer within 20 bases.
  • RNase: An enzyme found in cytoplasm of cells, which digest RNA NOT DNA.


References:

Journals

1. Ambros, V. et al.A uniform system for microRNA annotation. RNA.2003.9, 277-279.PMID: 12592000

2. Babak, T. et al.Probing microRNAs with microarrays: Tissue specificity and functional inference. RNA.2004.10, 1813-1819.PMID: 15496526

3. Britten RJ, Davidson EH. Gene regulation for higher cells: a theory Science. 1969. 25;165(891):349-57.PMID: 5789433

4. Buck, A. H. et al. Discrete Clusters of Virus-Encoded MicroRNAs Are Associated with Complementary Strands of the Genome and the 7.2-Kilobase Stable Intron in Murine Cytomegalovirus. Journal of Virology, 2007.81, 24, 13761–13770.PMID: 17928340

5. Calin GA, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002.26;99(24):15524-9.PMID: 12434020

6. Eckardt, N. A.Systematic Small RNA on the Move.2004.16, 1951–1954.PMID: 15258266

7. Frankel, L. B.Programmed Cell Death 4 (PDCD4) Is an Important Functional Target of the MicroRNA miR-21 in Breast Cancer Cells. J. Biol. Chem., 2008.283, 2, 1026-1033.PMID: 17991735

8. He, L.,Hannon,G.J., “MicroRNAs: small RNAs with a big role in gene regulation”. Nat Rev Genet. 2004;5(7):522-31.PMID: 15211354

9. Iorio, M. V. MicroRNA Gene Expression Deregulation in Human Breast Cancer. Cancer Res, 2005.65, 16, 7065-7070.PMID: 16103053

10. Cannell,I.G., Kong,Y.W.,Bushel.M., How Do MicroRNAs Regulate Gene Expression? Sci. STKE, 2007.367, 1.PMID: 19021530

11. Kim, H. K. et al.Muscle-specific microRNA miR-206 promotes muscle differentiation. The Journal of Cell Biology, 2006.174, 5, 677-687.PMID: 16923828

12. Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNAs in vivo with 'antagomirs'. Nature. 2005. 1;438(7068):685-9.PMID: 16258535

13. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001. 26;294(5543):853-8.PMID: 11679670

14. Latronico, M. V. G. et al. Emerging Role of MicroRNAs in Cardiovascular Biology. Circ Res., 2007.101, 1225-1236.PMID: 18063818

15. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science. 2001.26;294(5543):858-68.PMID: 11679671

16. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science. 2001.26;294(5543):862-4.PMID: 11679672

17. Lee, R.C., Feinbaum, R.L., and Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell.1993. 75, 843–854.PMID: 8252621

18. Loong, S. N. K. & Mishra, S. K. Unique folding of precursor microRNAs: Quantitative evidence and implications for de novo identification. RNA. 2007.13, 2, 170-187.PMID: 17194722

19. Maher, C. & Stein, L. & Ware, D. Evolution of Arabidopsis microRNA families through duplication events. Genome Research, 2006.16, 510-519.PMID: 16520461

20. Piriyapongsa, J. & Marin˜o-Ramı´rez, L. & Jordan, I. K. Origin and Evolution of Human microRNAs From Transposable Elements. Genetics, 2005.176, 1323–1337.PMID: 17435244

21. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000. 24;403(6772):901-6.PMID: 10706289

22. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. MicroRNAs in plants. Genes Dev. 2002.1;16(13):1616-26.PMID: 12101121

Book/s

23. Appasani,K.'MicroRNAs: From basic Science to disease biology'.Cambridge,(2008).pp 295-335.

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