RNAi – Is one suffix sufficient?

RNA-directed translational gene silencing in many organisms appears to represent a later evolved function for this versatile molecule, and the observation that protein domains similar to those of RISC complex components in Tetrahymena, fission yeast, fungi and archaeal bacteria suggests that RNA interference as it is currently understood may have developed as a functional entity in the last common ancestor of eukaryotes (Cerutti and Casas-Mollano, 2006). Crystal structures indicate that the PIWI domain of ARGONAUTE proteins is a member of the RNase H superfamily, supporting the notion that RNAi evolved as a defense against genomic parasites (Parker et al, 2004; Cerutti and Casas-Mollano, 2006). A range of new studies are now beginning to reveal both a diversity of cellular small RNAs, and potential functions other than translational silencing in higher organisms. It is becoming increasingly apparent that the role of RNA in coordinating and facilitating a wide variety of cellular processes has been underestimated. Traditional exogenous short RNA mediated gene silencing efforts have been designed to target an mRNA sequence. Most endogenous miRNA also appear also to target the transcribed RNA and so RNAi has generally been considered a post-transcriptional regulatory mechanism (Bernstein et al, 2001; Broderson and Voinnet, 2006). Experiments in fission yeast have showed that complexes containing siRNAs can be co-purified with proteins involved in transcriptional silencing, suggesting that small RNAs might have nuclear silencing roles also (Reinhart and Bartel, 2002; Volpe et al, 2002). Whilst transcriptional regulation by RNA has been implied since the observation of RNA-induced DNA methylation in plants over 10 years ago, (Wassenegger, 1994), the relevance of this observation to small RNAs and animal cells has only recently come into focus. Small RNAs play a role in histone H3 methylation at centromeres in S.pombe, indicating that such a mechanism is not limited to plants (Volpe et al, 2002; Reinhart and Bartel, 2002; Buhler et al, 2006). Experiments in C. elegans and Drosophila identified potential transcriptional regulatory roles for small RNAs that involve chromatin modification (Pal-Bhadra et al, 2004; Grishok et al, 2005). RNAi-induced Transcriptional Gene Silencing (RNAiTGS) in C. elegans is dependent upon heterochromatin factors, reduced by histone deacetylase inhibitors and is associated with a decrease in acetylated histone H4 protein and less polymerase II recruitment to the initiation complex. Mutations in the RNAi pathway genes dcr-1, rde-1, rde-4, and rrf-1 refract RNAi-TGS, strongly indicating that this is a nuclear mechanism of transcriptional regulation mediated by the RNAi machinery (Grishok et al, 2005). The experimental application of RNA interference has become routine, but several new observations suggest that small RNAs might induce unappreciated cellular effects such as sequence specific transcriptional silencing or activation. Twenty-one nucleotide dsRNA targeted to the promoter regions of stably transfected CCR5 and RASSF1A or endogenous Huntingtin, Androgen receptor or Progesterone receptor loci represses transcription in cell culture, whilst at the promoters of E-Cadherin, p21 and VEGF genes, exogenous dsRNA enhances the steady state level of transcription (Janowski et al, 2006; Kim et al, 2006; Li et al, 2006). Answering the question of whether these observations result from currently documented silencing mechanisms, or represent novel (perhaps endogenous) pathways of small RNA regulation is of some significance as, if borne out, the findings might offer a means to specifically activate transcription with small RNAs in an analogous manner to gene silencing by post transcriptional RNAi.