Gene silencing in the nucleus: mechanisms and new phenomena

Abstract

Silencing of homologous genes by exogenously introduced dsRNA was first observed in C. elegans. Endogenous small RNAs (siRNAs, piRNAs, miRNAs) mediate regulation of expression of genes post-transcriptionally or at the level of transcription, when argonaute proteins complex with small RNAs to target genomic loci for chromatin modifications in a sequence-specific manner, in the nucleus. There have been previous reports of regulation of expression by targeting mRNA for silencing in the cytoplasm. We identified a region in the genome, flp-17 locus that is amenable to nuclear silencing mechanisms in a wildtype animal. C. elegans exhibits strong anti-foreign genome silencing in their germline as defense against invading viral or transposon DNA. This activity is extended to transgene DNA, resulting in its silencing in the germline. Mos1 is a foreign element, a transposon from Drosophila that is heterologously inserted in C. elegans genome. Trangenes are integrated in the chromosomal DNA by Mos1 based Single Copy insertion technology where homologous regions flanking the gene of interest promotes recombination and thus its integration at a specific mos site on the worm genome. In our experiments we observed robust silencing in the somatic cells of transgenes that were intended to be integrated in ttTi5605 mos site on C. elegans genome by homologous recombination. The silencing phenomenon involves epigenetic mechanisms. We hypothesize that over several generations, the worm has “learned” that mos is a foreign element and when transgene is integrated at that site, it is amenable to silencing by the epigenetic machinery. Furthermore, we identified a previously undescribed mutation (yy14) in eri-6 gene and show evidences pointing to a role in silencing of transgenes in somatic tissues. We observed an increase in trans-spliced mRNA from eri- 6/7 genes in yy14 mutants. Our model reasons out that increase in trans-spliced mRNA results will lead to upregulation of small RNAs (26G RNAs) that efficiently function in the silencing of our transgene, which needs further verification. In addition, ABC transporters in C. elegans have been previously shown to be required for efficient RNAi. haf-2, haf-6 and haf-9 mutants also exhibit defects in transposon mobilization. Chromatin modification by epigenetic machinery prevents mobilization of transposable elements. This includes RNAi effectors like siRNAs, Dicer and argonaute proteins. Thus, a previous study in the lab, highlights a link between ABC transporters and RNAi mechanisms in C. elegans. It is important to characterize the dimerization pattern of half ABC transporter proteins to gain insights on their functions and precise roles in RNAi.