RNA Interference

RNA interference (RNAi) is a conserved gene regulation process initiated by small silencing RNAs that act in a sequence-specific manner. Fire and Mello first described this phenomenon in C. elegans in 1998. Early names for the phenomenon were post-transcriptional gene silencing (PTGS), transgene silencing, and quelling. It occurs in all eukaryotes, yeast to mammals. RNAi plays a role in gene expression regulation, transposon control, and protection of cells against viral infections. The two classes of small silencing RNAs include microRNA (miRNA) and small interfering RNA (siRNA).

miRNAs in RNAi

MiRNAs are small non-coding RNAs encoded by the genomes of plants and animals, as well as their viruses. They are 20-25 nucleotides long RNAs that regulate gene expressions by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs. The first miRNA, lin-4 was discovered by a team of scientists headed by Victor Ambros, in the worm C. elegans. The lin-4 gene did not code for any protein but formed short RNA that was complementary to target mRNAs preventing the translation of genes. Later on, in 2000, another miRNA, let-7, discovered by Gary Ruvkun's group also exhibited similar activity.

miRNAs are involved in developmental timing, cell differentiation, proliferation, cell death, and oncogenesis. The biogenesis of a miRNA starts in the nucleus by transcribing a long primary transcript (pri-miRNA) by RNA polymerase II. The pri-miRNAs are processed by Drosha, which is an RNase III enzyme, along with a dsRNA-binding protein, DGCR8 in mammals and Pasha in Drosophila, into 70-100 nucleotide hairpin structures called pre-miRNAs. These structures are exported to the cytoplasm by exportin-5 where they are further processed into a mature miRNA.

Dicer cleavage of the pre-miRNA takes place in cytoplasm, and the resulting miRNA duplex can be loaded into the RISC. Argonaute (Ago) proteins play a very crucial role within the RISC, and these proteins facilitate the involvement of miRNAs in gene silencing. The PAZ domain of Ago interacts with the 3' end of mature miRNA, and its PIWI domain is similar to ribonuclease-H, which is essential in gene silencing.

Plant vs. Animal miRNA Biogenesis

This process is different in plants compared to animals. Only one type of enzyme, DCL1, is capable of executing the function of Drosha and Dicer enzymes.

In addition, the plant miRNA duplexes are methylated by HEN1 before leaving the nucleus.

miRNA in gene silencing

Gene silencing by miRNAs may be brought about either through the degradation of mRNA or through the inhibition of translation, and this mechanism is dependent upon the degree of complementarity between the miRNA and its target mRNA. Where there is high complementarity, the mRNA may be degraded by Ago, whereas partial complementarity results in prevented translation. In most cases, target mRNAs are recognized by the "seed sequence," an important 2-8 nucleotide sequence at the 5' end of miRNAs.

RNAi mediated by siRNAs

In the siRNA-mediated pathway of RNA interference, dsRNAs are processed into siRNA duplexes by a Dicer enzyme. The siRNAs have around 21 nucleotides, along with two-nucleotide overhangs at the 3' ends. Dicer is a ~200 kDa multi-domain enzyme of the RNase III family. Dicer chops dsRNA into siRNAs, and packaged these with a dsRNA-binding protein into the RNA-induced silencing complex (RISC). The guide strand of the siRNA then hybridizes to the target mRNA by full complementarity so that RISC can silence the gene. The passenger strand, also called the anti-guide strand, was degraded during this activation. Argonautes are endonucleases which cleave the target mRNA at the complementary site.

Conclusion

The main difference between siRNAs and miRNAs is based on their origins. While siRNAs are derived from double-stranded precursors to RNA, miRNAs are derived from single, extended transcripts that assume the form of imperfectly base-paired hairpin structures. The siRNAs seem to generally have full complementarity to target mRNAs and cause cleavage to a specific target, whereas miRNAs usually have partial complementarity and degrade the translation of a number of mRNAs containing similar sequences.

PiRNAs

More recently, a class of small RNAs around 25-30 nucleotides in length was found to bind to the Piwi clade of Argonaute proteins. Unlike siRNAs and miRNAs, piRNAs are derived from single-stranded RNA precursors and do not require Dicer activity. They are predominately antisense and can be seen as sense and are 2'-O-methylated at their 3' termini, similar to siRNAs. piRNAs may be involved in the suppression of transposon expression either through cleavage of mRNA or by modifying the chromatin state at transposon loci.

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