MicroRNAs : Biogenesis and postpost-transcriptional regulation.

What is microRNA?

microRNA by current convention is defined as a ssRNA of 22 nt. in length. Generated by the endogenous RNAse-III type enzyme Dicer from an endogenous transcript containing a local hairpin structure. For any small RNA discovered by cDNA cloning to be classified as miRNA it should: 1. expression should be confirmed by hybridization to a size fractioned RNA sample usually by northern blotting. 2. small RNA sequences must be present in one arm of the hairpin precursor lacking internal loop or bulges. 3. small RNA sequences should be phylogenetically conserved

miRNA : Biogenesis Overview

Biogenesis: Overview

Is the biogenesis mechanism Universal to all miRNAs ??? And the answer to this question is NO. Maturation steps specific to individual miRNAs has been uncovered. Offer a plethora of regulatory options after transcription with multiple proteins effecting miRNA processing effeciency.

miRNA genes on the basis of their genomic location. miRNA genes can be grouped on the basis of their genomic location: Exonic miRNA in nc-transcription units. Intronic miRNA in nc- transcription units. Intronic miRNA in protein coding transcription units. Mixed miRNA can be designated to any of the above groups depending on the splicing pattern AND is not a separate group.

Nuclear processing by Drosha (RNASEN)

Pri-miRNA cleavage by Drosha-DGCR8 microprecessor complex endonucleolytically. Drosha is 160 Kda protein and binds to DGCR8/Pasha (120KDa). Drosha has two RNAseIII Domain and a dsRBD. DGCR8/Pasha has 2 dsRBDs which stably interact with pri-miRNA and functions as molecular ruler determining the precise cleavage site. The two RNAse domain cleave the 5 and 3 arms of pri-miRNA hairpin. Drosha cleaves 11bp from the ssRNA/dsRNA junction at the base of hairpin stem.

Export to the cytoplasm

After nuclear processing pre-miRNA is exported to the cytoplasm by Exportin-5 (XPO-5) in complex with Ran-GTP. Members of the nuclear export receptor family bind cooperatively to a cargo as well as to the GTP-bound form of the cofactor Ran in the nucleus. Following export, GTP-GDP hydrolysis results in the release of cargo from the export complex.

An insight into Dicer

Apart from two RIIIDs & a dsRDB, Dicer has a long N-terminal segment that contains a DEAD-BOX RNA HELICASE DOMAIN, DUF283 Domain and a PAZ Domain. PAZ Domain is also found in ARGONAUTE PROTEINS. PAZ Domain binds to the 3 protuding end of small RNAs. Role of other domains remain unclear.

microRNA biogenesis in Plants

Drosha dependent processing model applies only to animal cells. Plant miRNA precursors are quite diverse and the stem-loops are usually longer than animal pri-miRNAS. DCL-1, one of the 4 Dicer Like Proteins in A.thaliana is a nuclear protein and indicates that the mature 22 nt. miRNA might be generated in the nucleus in plants. Atleast one study shows that plant miRNAs, similar to animal miRNAs might be processed in a stepwise manner, in which DCL-1 mi9ght be responsible for all processing steps. HASTY(HST) is a plant homolouge of exportin-5.

Unwinding of miRNA duplex into guide and passenger strands. Following the cleavage by Dicer, the duplex is unwounded by the helicases e.g. p68, p72, RNA Helicase A (RKA) etc. preceded by the ouster of Dicer and its associated proteins, TRBP/PACT to form the active miRNP S or miRISC. The generation of guide strand depends on the thermodynamic stability of the base pairs at the two ends of the duplex: the miRNA strand with the less stable base pair at its 5 end in the duplex is loaded into RISC. RISC or miRNP comprises of miRNA, AGO2 and certain other proteins functioning as miRNP assembly or regulatory factors.

miRNA mediated post-transcriptional gene repression:

Conclusion

microRNA biosynthesis can no longer be viewed as one general pathway universal to all miRNAs. These specific differences in miRNA processing suggests multiple opportunities for post-transcriptional regulation of miRNA expression. Because little is known about the stability and degradation of miRNAs, this is a promising area for the discovery of novel regulatory mechanisms. It will be crucial to understand the regulation of miRNA function through modulation of the activity of RISC components and associated factors, through phosphorylation and other protein modifications In the near future therapeutic approaches can be developed based on smallRNAs targeting genes with an established disease association, like oncogenes.

References

Filipowicz, W., et. al. Mechanism of post-transcriptional regulation by microRNAs: are the answers in sight? Nature 9, 102-114 (2008). Winter, J., et.al. Many roads to maturity: microRNA biogenesis pathway and their regulation. Nature 11, 228-234 (2009). Kim, N.V., MicroRNA Biogenesis: Coordinated Cropping and Dicing. Nature 6, 376-385 (2005). Pillai, R.S., et.al. Repression of protein synthesis by miRNAS: how many mechanisms? TRENDS in Cell Biology 17, No.3, 118-126 (2007).

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