RNA Interference and RNA Silencing

Barford Group

Section: Section of Structural Biology

RNA silencing mechanisms mediated by small RNAs regulate gene expression at both post-transcriptional and transcriptional levels. Currently the most extensively characterised system is targeted mRNA degradation mediated by small interfering RNAs (siRNAs). siRNAs are 21-25 nucleotide double-stranded RNAs with 5' phosphate groups and 3' dinucleotide overhangs. siRNAs are produced from double-stranded RNAs (dsRNAs) through the action of the RNase III enzyme Dicer. This process may have evolved as a cellular defence response to viral infection or the activity of transposable DNA elements. Related to the action of siRNAs are the mechanisms of silencing mediated by microRNAs (miRNAs). miRNAs closely resemble siRNAs in size and structure. miRNAs are, however, encoded as stem-loop precursors in the genome, which are then processed through the actions of the RNase III enzyme Drosha together with Dicer. Many miRNAs regulate gene expression by binding and inhibiting the translation of mRNAs, although in some instances miRNAs can also target mRNAs for destruction. The mechanistic differences between siRNAs and miRNAs can be attributed partly to the degree of complementarity between the short RNA and its target. A third mechanism of silencing operates at the transcriptional level, by targeting chromatin remodelling factors to heterochromatic regions. As with the siRNA and miRNA pathways, the specificity of nucleic acid targeting (perhaps this time DNA), is mediated by small RNAs.

siRNAs (and many miRNAs) operate in large ribonucleoprotein complexes termed RISC (RNA-Induced Silencing Complex). RISC assembly requires ATP and proceeds via a series of intermediate sub-complexes. During this process, the double-stranded siRNA is unwound to incorporate only a single-stranded RNA molecule in the final active complex. The relative stabilities of the two ends of the double-stranded siRNA appear to determine which strand becomes incorporated: siRNAs are functionally asymmetric. Once assembled, siRNA-RISC functions as a multiple-turnover complex that recognises and cleaves mRNA strands complementary to the incorporated single-stranded siRNA. The mRNA is cleaved between the nucleotides paired to bases 10 and 11 of the siRNA - the RISC catalytic site appears therefore to be fixed relative to the binding position of the 5' end of the siRNA. Cleavage is magnesium-dependent and yields 3' hydroxyl and 5' phosphate groups on the resulting mRNA strands.

As mentioned above, the method of miRNA-directed translational inhibition shares many similarities with siRNA-directed mRNA cleavage. It is proposed that for RISC to catalyse mRNA cleavage, a contiguous A-form helix must be formed between the short guide RNA and the mRNA target in the region of the scissile bond. Presumably this is required to generate the correct reaction geometry in the active site. This would then be sufficient to explain the observed mechanistic differences between perfectly complementary siRNAs and imperfectly complementary miRNAs.

The function of short RNAs in chromatin remodelling is understood principally from studies in the yeast S. pombe. Orthologues of components of the siRNA/miRNA systems are required for methylation of lysine 9 on histone H3 (and thus the recruitment of the crucial remodelling factor HP1), gene silencing in heterochromatin and proper centromere and telomere function. However, the effector complex in this case appears to be distinct from RISC. Small RNAs are incorporated instead into a complex termed RITS (for RNA-induced Initiation of Transcriptional Gene Silencing). It is proposed that the RITS complex, guided by the small RNAs, localises remodelling factors to their sites of action. Recent genetic studies have uncovered potentially similar systems in Arabidopsis, Drosophila and mammals.

Studies of Argonaute Proteins

Argonaute proteins have been implicated by genetic and biochemical methods in all the mechanisms involving small RNAs discussed above. A striking finding is that Argonaute family members are the only proteinscommon to all RISC-related complexes and the RITS complex. Indeed, it has been demonstrated recently that an Argonaute protein is the only polypeptide present in a highly purified active form of Drosophila RISC. This suggests that Argonaute proteins possess unique functions with regard to small RNAs involved in RNA interference. Argonaute proteins are characterised by two domains: a ~20 kDa N-terminal PAZ domain and a ~40 kDa C-terminal PIWI domain. Until recently, however, the biochemical functions of Argonaute proteins have remained obscure. A breakthrough was achieved with the determination of the structure of the N-terminal PAZ domain found in all Argonaute proteins. This structure suggested a role for PAZ in RNA binding, a proposal subsequently confirmed by the solution of a structure of PAZ in complex with an siRNA-like RNA helix. This structure demonstrated that PAZ does indeed interact with RNA, and moreover that PAZ may serve as the binding module for the end of short dsRNAs and as the anchor for the 3' end of short ssRNAs in RISC, and perhaps RITS. Phylogenetic analysis of eukaryotic Argonaute proteins delineates two subfamilies (Piwi and Ago), resembling Drosophila Piwi and Arabidopsis Ago1, respectively. At present, no broad functional distinction exists between these two subfamilies.

We determined the crystal structure of a Piwi protein (AfPiwi) from the archaean Archaeoglobus fulgidus (Parker et al., 2004). The protein features a prominent positively charged channel reminiscent of RNA binding proteins, and we demonstrate that AfPiwi forms a distinct complex with an siRNA-like RNA duplex. The identification of an RNase H-like fold within the C-terminal portion of the PIWI domain, together with the mapping of conserved residues onto the AfPiwi structure allows us to propose a mechanism for RISC-mediated RNA cleavage. We identified two conserved regions in the molecule. The first set of residues cluster to a region of AfPiwi resembling the RNase H active site, suggesting that in eukaryotic Argonaute proteins this region may possess RNase activity. The second cluster of conserved residues, which coordinates a metal ion, and is rich in basic residues, is positioned within a pocket ~20 Å from the putative RNase H-like catalytic site. The distance between these two sites matches the dimensions between the obligatory 5’phosphate of the guide RNA and the scissile phosphate between the nucleotides paired to bases 10 and 11 of the guide strand. These findings provide a molecular explanation for RISC-mediated mRNA cleavage.

In recent work, we have determined the structure of a complex of AfPiwi in complex with a 16 nucleotide siRNA mimic. This structure (Parker et al., 2005) provides a model of an argonaute protein in complex with a guide RNA bound to target mRNA. The structure reveals a 5’ guide binding pocket formed from CR1 that anchors the 5' end of the guide strand to the PIWI domain. 

Argonaute publications:

J.S. Parker, S.M. Roe and D. Barford (2005). Structural insights into mRNA recognition from a PIWI domain – siRNA guide complex.  Nature, 434, 663-666

Parker JS, Roe SM, Barford D. (2004). Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity.  EMBO J., 23, 4727-4737