Small RNA Biogenesis and Role in Gene Expression Regulation - Essay Example

Topic: Small RNA biogenesis and role in gene expression regulation Instructions: The essay should be typed, double-spaced and be up to 10 pages in length including 6-7 pages of text, 2-3 pages of figures and tables which can be reproduced from the original literature, and 1 page for bibliography which could contain 5-10 references (I will upload the assigned material to my personalised control panel, but please find more references to complete the requirement). Instructions files attached: 1. Argonaute proteins key players in RNA silencing.pdf 2. Let Me Count the Ways Mechanism of Gene Regulation by miRNAs and siRNAs.pdf 3. miRNAs Get an Early Start on Translational Silencing.pdf 4. Multi-tasking Argonautes.pdf Created: 2008-03-31 10:51 Deadline: 2008-04-07 09:04 Time Left: 162 hours Style: Harvard Language Style: English (U.S.) Grade: n/a Pages: 9 Sources: 10 ESSAY Small RNA’s are a large family of regulatory molecules with diverse functions 4. They are noncoding RNAs, encoded in both prokaryotic and eukaryotic genomes 1, 3 and are involved in a various cellular processes in the cytoplasm and in the nucleus 2. Their function is primarily regulatory in nature ranging from translation, message stability, RNA degradation and signaling epigenetic modifications in DNA and histones 1, 2, 5. The process by which small RNAs target mRNAs for degradation is now commonly referred to as RNA interference or RNAi 2. They regulate post-transcriptional modifications from bacteria to mammals and are also implicated in the regulation of critical pathways such as stress response in bacteria, developmental timing and cell differentiation in plants and metazoans 3, 5. They also play a role in antiviral defense 5. The small RNA’s are of two types, micro RNA’s (miRNAs) and small interfering RNA’s (siRNAs) 7 and the in their effective state they range from 21 to 25 nucleotides 5, 6. Both types of small RNAs are produced from longer RNA precursors with the help of nucleases called DICER in animals and DICER LIKE (DCL) in plants 7. The siRNA and miRNA have different origins. The miRNAs (approximately 21 to 22 nucleotides) are encoded by their own genes 7 and are generated from the non-protein coding dsRNA region of the hairpin shaped precursors 1, 5. The siRNAs (approximately 21 to 24 nucleotides) are derived from long double-stranded RNAs 1. They are processed from these dsRNA precursors that arise from mRNAs, transposons, viruses or heterochromatic DNA 5, 7. Biogenesis of small RNAs miRNA They were discovered initially in nematodes in 1993. They are found in plants and animals and in the viruses that infect them. They play a vital role during gene expression during plant and animal development and it has been postulated that they regulate one-third of human genes 9. The coding sequences of most miRNAs lie in the genome, distinct from the protein coding sequences, while most of the mammalian miRNA genes lie within the introns of protein coding genes or in the untranslated regions of protein coding genes or within the introns or exons of non-coding genes. The loci coding for different miRNAs may lie distant from each other or clustered thereby sharing similar expression patterns 9. Their biogenesis involves the formation of an initial precursor transcript or primary mRNA (pri-mRNA) which is followed by nucleolytic processing steps 5. In animals the maturation of miRNAs requires the pri-miRNA to adapt a fold back structure 5, 7 to facilitate cleavage by a ribonuclease III (RNase III) termed Drosha, in the presence of a dsRNA binding domain protein. This process takes place in the nucleus. This cleavage results in a  70 nucleotide stem loop pre-miRNA which is exported to the cytoplasm, by a protein called exportin, where it is acted upon by a second RNase III termed DICER to give a mature  21 nucleotide miRNA duplex 9, 10, 13. These have a free 2’ 3’ hydroxyl group. In plants, which lack a Drosha homolog, a RNase III termed DICER LIKE1 (DCL 1), localized in the nucleus is involved in both the cleavages of the miRNA. The mature miRNA is then exported from the nucleus. These have a methyl group on the ribose of the last nucleotide 9. Arabidopsis contains 4 DCL genes (DCL 1 to DCL 4) 5, 7 and DCL 1, DCL 2 and DCL 3 have been shown to be involved in miRNA biogenesis 7. fifteen classes of miRNAs have been identified in Arabidopsis 7. Zamore D. P, Du T, 2005, The miRNA biogenesis pathway. (A) Animal and (B) plant miRNA biogenesis. Mature miRNAs are indicated in red, whereas the miRNA* strands are in blue, Development, The Company of Biologists, viewed on April 4, 2008. siRNA These are derived from large double stranded RNA precursors 6, 7. there are three classes of endogenous small interfering RNAs, transacting (ta-siRNA), heterochromatic (hc-siRNA) and natural antisense (nat-siRNA)13all of which are processed from existing double stranded RNAs 10. They are chemically similar to miRNA and their size ranges between 21 and 24 nucleotides 5. The endogenous siRNAs precursors in plants include retroelements, transposons, other repeated sequences, psudogenes, intergenic regions and a few expressed genes. Exogenous siRNAs can arise from sense and hairpin transcript forming transgenes and by viruses. siRNA precursors can also be formed by the activity of cellular RNA dependent RNA polymerases 5. The Arabidopsis plants contains at least three active RdRp genes, termed RDR1, RDR2 and RDR6 5. These long perfectly paired dsRNAs are processed by Dicer, a. RNAse III enzyme, which cleaves one phosphodiester bond on opposite sides of the substrate giving rise to a 21 – 28 nucleotide long double stranded RNA, with a 5’ phosphate and 2’3’ hydroxyl termini 10. Trans-acting siRNAs are plant specific RNAs, and their maturation involves miRNAs. Hutvagner G, Simard J.M, 2008, Diverse sources of small RNA molecules, Nature Reviews Molecular Cell biology, Vol 9, viewed on April 7, 2008. Formation of the RISC Complex required for their regulatory role The mature siRNA and miRNA assemble into an effector complex; the RNA induced silencing complex (RISC) 10, which is the active regulatory complex that performs small RNA induced transcript degradation 7. The assembly of RISC is asymmetric, as one strand of siRNA or miRNA preferentially incorporates into a protein RNA complex 9, 10, 11. The strand, in the duplex which has less thermodynamic stability in base pairing near its 5’ end serves as the template for RNA silencing, while the other strand is degraded 6, 11. The RISC complex studied in flies is composed of the Dicer Dcr2, which is involved in the processing of dsRNA into siRNA and R2D2, an RNA binding protein, both of which bind to the double stranded siRNA. The R2D2 protein binds to the thermodynamically favorable strand of the siRNA and it positions Dcr2 at the opposite end of the duplex 10. In addition this protein, RISCs also contain the unique and highly conserved Argonaute proteins (AGO) 5, 9, 10, 11, as the Dcr2/DRD2 complex cannot unwind the target dsRNA on their own 10. These proteins by binding to the small dsRNAs, control protein synthesis, affect messenger RNA stability and also participate in the production of Piwi interacting RNAs 11. It contains a PIWI domain, which has RNase H like activity that cleaves RNA using a DNA template thereby functioning as the catalytic core of the protein 9, 11. However, in contrast to plant miRNAs, the catalytic function of the AGO protein, is not required during miRNA mediated gene repression in animals. Hutvagner G, Simard J.M, 2008, Assembly of the Argonaute- Small RNA complex, Regulatory roles of small RNAs. Both miRNA and siRNA repress the target mRNAs by interfering with translation or by guiding processes that are involved in mRNA degradation. However, the mRNAs regulated by miRNAs largely remain unknown 8. Translation Repression In a study carried out with rabbit reticulosyte lysate system 8, it was demonstrated that miRNA mediated translation repression required a functional m7 G cap and a poly- A tail. The AGO proteins in the miRNA bind to the cap which releases the initiation factor thereby inhibiting translation 8, 12. A cap independent repression mechanism has also been proposed 12. Other proteins involved in the repression process are TNRC6 (required in Drosophila), MOV10/Armitage (functions as a RNA helicase), and RCK/p54 (implicated in miRNA independent translational silencing.) 12. Meister G, 2007, mi-RNA guided repression of translation initiation. Cell, 131, Elsevier Inc, Viewed on April 7 2008. The repressed mRNA protein assemblies (mRNPs) aggregate into larger structures called as pseudo- polysomes, which can disintegrate and active translation can be reinitiated 8. further it has also been suggested that miRNAs might also be involved in regulation of translation elongation 8. Translation activation The miRNA can activate translation under conditions like cell cycle arrest or treatment with replication inhibitors. This process will also require base-pairing, the exact mechanism of which has not yet been determined 12. mRNA Degradation There are two methods of degradation of mRNA by the small RNAs depending on the complementarity’s between them and the target mRNA. Direct endonucleolytic cleavage by AGO protein, of the target RNA takes place in case of full complementarity, which results in complete decay of the target. In case of incomplete complementarity, miRNAs accelerate mRNA turnover by removing the partially complementary poly A tail. This process coupled with the loss of poly A binding protein causes 5’ dacapping. This paves way to exonucleolytic cleavage. A. mRNA undergoing endonucleolytic cleavage by AGO, guided by a fully complementary siRNA and miRNA. B. mRNA undergoing poly A removal by deadenylase as directed by a partially complementary mi RNA. Wu L, Belasco G.J, 2008, Mechanisms by which siRNAs and miRNAs trigger mRNA decay. Molecular Cell 29, Elsevier Inc. Viewed on April 7, 2008. RNA silencing pathways. The Argonaute protein that binds to the small RNAs, forming the functional RISCs are chiefly involved in RNA silencing. Through its PAZ domain, the AGO protein binds to the 3’ end of the thermodynamically favorable stand of siRNA or miRNA, through interaction with the Dcr2 9, 11. Once bound to the small RNAs, the AGO protein will cleave the passenger or the thermodynamically non-preferred strand non active strand, thus initiating unwinding to generate the active RISC of the Argonaute and small RNA 9, 10, 11. This is similar to the way it cleaves the target dsRNA 10, 11. The selection of the strand depends on the thermodynamic stability of the 5’ end of the small RNA 11. Other regulatory activities of small RNAs A recent study has proposed that miRNAs play significant roles in plant development like the regulation of flowering time and floral organ identity. They have also been thought of as a suitable candidate for systemic silencing signal 7. Recent studies indicate that miRNA are involved in biotic and abiotic stress response in plants. In a study the expression of a particular miRNA was increased upon sulphate starvation, thereby indicating that they can be induced by environmental factors 7, 13. The nat- siRNA has also been proposed to fuction in stress adaptation 13. Small RNAs have the ability to regulate the expression of multiple target genes. They are implicated in the regulation of oxidative response, osmotic response, acid response, quorum sensing, SOS response to DNA damage, glucose-phosphate stress and many more 3. Reference: 1. Pichon, C 2005, Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains, PNAS, Vol 102, no 40, viewed on April 7, 2008. 2. Meyer, P 2005, Plant Epigenetics Annual Plant Reviews, Vol 19, Blackwell Publishing. Viewed on April 5, 2008. 3. Levine E, Zhang Z, Kuhlman T, Hwa T , 2007, Quantitative Characteristics of Gene Regulation by Small RNA. PLoS Biol 5(9): e229, viewed on April 7, 2008. doi:10.1371/journal.pbio.0050229 4. Kim, NV, 2006, Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes, Genes and Development 20, Cold Spring Harbour Laboratory Press, viewed on April 7, 2008. 5. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, et al. (2004) Genetic and Functional Diversification of Small RNA Pathways in Plants. PLoS Biol 2(5): e104, viewed on April 1, 2008, doi:10.1371/journal.pbio.0020104 6. RNAi fundamentals, 2006, Gene Expression, viewed on April 7, 2008, http://www.gnxp.com/blog/2006/06/rnai-fundamentals.php# 7. Eckardt, AN, 2004, Small RNA on the Move, The Plant Cell, 16, 1951-1954, American Society of Plant Biologists, viewed on April 2, 2008. http://www.plantcell.org/cgi/reprint/16/8/1951 8. Meister, G, 2007, miRNAs Get an Early Start on Translational Silencing, Cell, 131, Elsevier Inc. viewed on April 3, 2008. 9. Du, T, Zamore DP, 2005, microPrimer: the biogenesis and function of microRNA, Development 132, 4645-4652, The Company of Biologists. Viewed on April 4, 2008. 10. Hutvagner, G, 2005, Small RNA asymmetry in RNAi: Function in RISC assembly and gene regulation, FEBS letters 579, 5850-5857, viewed on April 7, 2008. 11. Hutvagner, G, Simard, JM, Argonaute proteins: key players in RNA silencing, Nature Reviews, Molecular Cell Biology, Vol 9, Viewed on April 7, 2008. 12. Wu, L, Belasco, GJ, Let Me Count the Ways: Mechanisms of Gene Regulation by miRNAs and siRNAs, Molecular Cell, 29, Elsevier Inc. viewed on April 7, 2008. 13. Philipps, RJ, Dalmay, T, Bartels, D, 2007,The role of small RNAs in abiotic stress, FEBS letters, viewed on April 7, 2008. Read More