RNA INTERFERENCE (RNAi)- Discovery,Mechanism

CONCEPT OF RNA INTERFERENCE (RNAi):

RNA interference (RNAi) is one of the most exciting and fascinating discoveries of the past decade in functional genomics. RNAi is rapidly becoming an important method for analyzing gene functions in eukaryotes and holds promise for the development of therapeutic gene silencing. RNA interference is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (TGS) or by activating a sequence- specific RNA degradation process [PTGS/RNA interference (RNAi)] . RNA interference (RNAi) is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. The first evidence that dsRNA could achieve efficient gene silencing through RNAi came from studies on the nematode Caenorhabditis elegans. Further analysis in the fruit fly Drosophila melanogaster have contributed greatly toward understanding the biochemical nature of the RNAi pathway.

RNAi is a particularly effective biotechnological research technique for identifying the functions of genes . RNAi causes the silencing of gene expression within the cell by introducing double stranded RNA which is homologous to the target site of the gene. The gene silencing generally results not only from the inhibition of transcription through cleavage and degradation of target mRNA, but it also results from inhibition of protein synthesis by blocking translation of the intact mRNA. RNAi is a powerful tool because of its specificity, efficiency and potency to knock down any specific gene of interest. The marked difference between this technique, and that of "gene knockout", is that the gene remains present and completely unmodified, the only true effect is in the prevention of translation of messenger RNA (mRNA). During knockout, it is the gene itself that is acted upon.

Discovery of RNAi:

Molecular biologists had applied various methods such as insertion of TDNA elements and transposons, treatment with mutagens or irradiation and antisense RNA suppression to knockout gene expression at the mRNA level for the last few years prior to the discovery of RNAi. Apart from being time consuming, the above methods did not always work satisfactorily. This background lead to the discovery of a novel phenomenon called as RNAi. 

Napoli and Jorgensen were the first to report an RNAi type of phenomenon in 1990 . The goal of their studies was to determine whether chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, was the rate-limiting enzyme in anthocyanin biosynthesis. The anthocyanin biosynthesis pathway is responsible for the deep violet colouration in petunias. In an attempt to generate violet petunias, Napoli and Jorgensen over expressed chalcone synthase in petunias, which unexpectedly resulted in white petunias. The levels of endogenous as well as introduced CHS were 50-fold lower than in wild-type petunias, which lead them to hypothesize that the introduced transgene was “co suppressing” the endogenous CHS gene.

Romano and Macino, (1992) also  reported a similar phenomenon in Neurospora crassa noting that introduction of homologous RNA sequences caused “quelling” of the endogenous gene.

In the year 1998, Andrew Fire and Craig Mello postulated the mechanism of RNA interference by introducing the exogenous dsRNA into the C. elegans. Both were awarded Nobel prize in the category of physiology or medicine in 2006 .

Mechanism of RNAi:

Mechanism of RNA interferences as understood is that it comes into play when a double stranded RNA is introduced either naturally or artificially in a cell. An endoribonuclease enzyme cleaves the long dsRNA into small pieces of RNA. The small pieces could be mi RNA or si RNA depending upon the origin of long dsRNA i.e. endogenous or exogenous respectively. A double stranded RNA may be generated by either RNA dependent RNA polymerase or bidirectional transcription of transposable elements or physically introduced. The mechanism of RNA interference can be divided into 2 stages: 1st Initiation, 2nd Effector.

Initiation:

Acoording to many researchers this stage is characterised by generation of siRNA mediated by type III endonuclease Dicer. In Drosophila, Dicer, which is a large multidomain RNase III enzyme has been identified in existence into two forms: Dcr-1/Loquacious (Loqs)-PB (also known as R3D1-L[long]): generates miRNA and Dcr-2/R2D2 generates siRNA. Dcr-1 share a structural homology with Dcr-2, despite that they display different sets of properties such as ATP requirements and substrate specifications . 

Dcr-1 is an enzyme that show ATP independent functions and affinity towards stem-loop form of RNA (precursor of miRNA) . It has been identified that Dcr-1 requires a double stranded RNA binding protein partner. In Drosophila Dicer-1 is seen to interact with the dsRBD protein Loquacious (Loqs). Immunoaffinity purification experiments reveal that Loqs resides in a functional pre-miRNA processing complex, and stimulates and directs specific pre-miRNA processing activity. These results support a model in which Loqs mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs ). 

Loquacious protein is supposed to be composed of three dsRNA binding domains, which is encoded by loqs gene into two types of proteins PA and PB; isoform to each other, by alternate splicing, among which PB is known to enhance the affinity of Dcr-1 towards pre-miRNA .

Dcr-2 shows ATP dependent activity with substrate specificity to double stranded RNA. Structurally homologous to Dcr-1, it also requires a double stranded RNA binding protein, namely R2D2, which function in association with specific RNase enzyme Dcr-2 forming a heterodimeric complex. R2D2, unlike Loq is supposed to be composed of two dsRNA binding domains  that interact with long double stranded RNA but does not regulate si RNA generating activity of Dcr-2 ). 

Studies of the physiological functions of R2D2 by the design of mutant strain have shown that R2D2 protein plays an important in development. It has also been shown to be responsible for the stability of Dcr-2 and decrease in the concentration of R2D2 in cells reduces the concentration of Dcr2 and vice versa. These results indicate that both ds RNA binding domains of Dcr-2 and R2D2 are critical for Dcr-2/R2D2 complex to bind and load siRNA into siRISC complex . Dcr-2 contains an RNA helicase domain, a DUF283 domain, and a PAZ domain at the N terminus as well as tandem RNase III motifs and a dsRBD motif at the C terminus.

It is unclear which of these domains physically contact siRNA. Since neither Dcr-2 nor R2D2 bind siRNA alone, it is possible that siRNA is bound at the interface between Dcr-2 and R2D2, which triggers a conformational change in either or both proteins, allowing them to bind siRNA co-operatively .

Effector stage:

The second stage of RNAi mechanism involves guide strand (duplex of siRNA and miRNA) incorporation. Guide strands are single strand duplexes of the miRNA and siRNA, and are integrated into effecter complexes that are involved in RNA silencing. These effecter complexes include the RNA-induced initiation of transcriptional gene silencing (RITS) or the RNA-induced silencing complex (RISC). The core of these RNA silencing effecter complexes, RISC was found to be composed of PPD Proteins (PAZ PIWI Domain proteins) which are highly conserved super-family. Members of PPD proteins contains centrally located PAZ (100 amino acids) and C-Terminal located PIWI (300 amino acids). 

The RNA interference model has taken a shape as; Dcr-2/R2D2 binds with siRNA, it forms RDI complex (R2D2 Dcr Initiator complex). RDI orients siRNA duplex so that the 5 prime end with lower melting temperature preferentially interacts with Dcr-2 the less stable end, R2D2 binds the more stable end of siRNA. This leads to thermodynamic asymmetry which helps the separation of guide strand from the passenger strand (other strand). Studies have shown that small change in the structure of siRNA effects on determination of the guided strand of the RISC complex which concludes, enzyme that governs the RISC assembly selects the siRNA strand incorporated into RISC on the basis of structure . Also the orientation of Dicer2/R2D2 on siRNA determines the guide strand. It suggests that structure siRNA determines the orientation of Dcr2/R2D2 which in turn determines the strand that will be incorporated into RISC as guide strand. 

Ago2-dependent complex, pre-RISC, was identified and Ago2 was found to be responsible for conversion of pre-RISC to holo-RISC i.e. the effective removal of passenger strand. The pre-RISC complex contains duplex siRNA whereas holo-RISC contains just the guide strand of the siRNA, the passenger strand having dissociated from holo-RISC. 5 prime end of siRNA acts as a target recognition guide which is required for the efficient RNA interference mechanism . Here the PIWI domain of the catalytic subunit of RISC (holo-RISC), Ago-2, acts as RNase H (highly conserved in Eukaryotes) and cleaves the target m-RNA backbone.

Transcriptional gene silencing:

It is the mechanism of gene silencing which is accomplished before the transcription of mRNA and is facilitated through chromatin remodeling and DNA methylation . The small RNAs produced by DCL cleavage can induce silencing both at the post-transcriptional level as well as at the transcriptional level. The mechanism of TGS is mediated by small RNA (24 ntds) produced by DCL3 cleavage of dsRNA which often uses RDRP (RNA dependent RNA polymerase). Chromosomal modifications such as DNA methylation at cytosine residues and histone methylation at specific lysine residues also induces the gene silencing through TGS because excessive methylation causes heterochromatization (Alberts et al., 2002). Sometimes, methylation of promoters causes chromosome remodeling thereby inhibiting the transcriptional factors to be attached at the promoters . It has been reported that the magnitude of DNA methylation is found to be more in the matured meristematic cells as compared to the younger diving cells thus, clearing that DNA methylation is involved in aging process in Pinus radiata . 

The deeply stained region of DNA are supposedly genetically inactive and very much condensed which is due to the ubiquitination, phosphorylation, methylation and acetylation of core histone proteins (H2A, H2B, H3 and H4) which are deeply involved in gene regulation (Lippman and Martienssen, 2004). When there is chemical changes in the tails of histones proteins, it generates a signal which regulates the accessibility of cells transcriptional machinery to the DNA through chromatin remodeling .

According to certain researches carried out in Arabidopsis indicates and Schizosaccharomyces pombe, these iRNA generate the signal for DNA methylation which is transmitted from cytoplasm to the nucleus . On the other hand, RNAi effector complex termed RNA induced Initiation of Transcriptional gene Silencing (RITS) required for heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) .

TERMINOLOGY:

Gene Knockout: It is a genetic technique in which one of an organism's genes is inactive or inactive expression of gene to study a function of loss gene. 

Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) about 20-25 nucleotides long Argonaute: Argonaute proteins are the catalytic components of the RNA induced silencing complex (RISC), the protein complex responsible for the gene silencing.

RISC: RNA-induced silencing complex, or RISC, is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA) which activates RNase and cleaves the RNA.

RNA helicase: exhibit helicase activity which separating two annealed RNA strands.