RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing

Abstract

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.Heterochromatic chromosomal domains occur in a wide range of eukaryotes and have a crucial role in regulation of gene expression, sister chromatid cohesion and maintenance of genomic stability 1,2 . The genome of the fission yeast Schizosaccharomyces pombe contains large blocks of heterochromatin associated with pericentromeric repeats, telomeres and the silent mating-type region (mat) 2 . Heterochromatin assembly at these loci involves a conserved array of histone modifications that includes histone deacetylation and the Clr4-mediated methylation of histone H3 at Lys9 (H3-Lys9), which is essential for the recruitment of chromodomain protein Swi6, a homolog of the fruit fly and mammalian HP1 proteins [3][4][5] .The mechanisms that specify particular chromosomal regions as sites of heterochromatin assembly are not known. It is well known, however, that the main targets of heterochromatin assembly are DNA repetitive elements such as transposons and satellite repeats present at pericentric regions in large eukaryotic genomes 2,6,7 . The RNA interference (RNAi) pathway 8,9 might be involved in heterochromatin nucleation at repeat loci 10-15 . In S. pombe, deletion of components of the RNAi pathway such as Argonaute (Ago1), Dicer (Dcr1) or RNA-dependent RNA polymerase (Rdp1) disrupts heterochromatinmediated silencing that correlates with loss of H3-Lys9 methylation and Swi6 association with heterochromatic loci 14,15 . In addition, siRNAs corresponding to centromeric repeats have been identified 16 , and the centromere-homologous cenH sequence (96% similar to dg and dh centromeric repeats 17 ) found at the mat locus is an RNAidependent heterochromatin nucleation center 14 . An RNAi effector complex (RITS) containing Chp1, Tas3 and Ago1 is involved in heterochromatin assembly 18 . RITS contains siRNAs that are believed to serve as specificity determinants for targeting RNAi effector complexes to homologous sequences 8,[...

Drosophila PIWI associates with chromatin and interacts directly with HP1a

“…The colocalization of PIWI and HP1a in pericentric heterochromatin may indeed reflect a simple relationship between the piRNA pathway, histone modification, and heterochromatin formation similar to the S. pombe system wherein AGO1-mediated TGS locally targets H3K9 methylation to create a binding site for the HP1 homolog Swi6 (Volpe et al 2002;Noma et al 2004). In Drosophila, our data on the specific interaction between PIWI and HP1a raise the possibility of an alternate pathway to HP1a-mediated heterochromatinization: A PIWI-piRNA complex might directly recruit HP1a to piRNA-corresponding genomic sequences, which could then recruit HMTs such as SU(VAR)3-9 to effect nucleation/spreading.…”

Cell Biology of the Arabidopsis Nuclear siRNA Pathway for RNA-directed Chromatin Modification

“…In fission yeast, RNAirelated targeting of heterochromatin is apparently initiated by bidirectional transcription, leading to the production of dsRNAs that can be diced. Resulting siRNAs are then stably bound to AGO1 within a protein complex known as RITS (RNA-induced initiation of transcriptional gene silencing) (Noma et al 2004;Verdel et al 2004), which also includes a chromodomain protein, Chp1, and a third protein of unknown function (Tas3). The siRNAs are thought to guide the complex to complementary RNA transcripts from a template-engaged DNA-dependent RNA polymerase (Grewal and Moazed 2003;Buhler et al 2006) which appears to be pol II (Djupedal et al 2005;Kato et al 2005).…”

miR-93/Sp7 function loop mediates osteoblast mineralization

“…(1,2) miRNAs negatively regulate translation of specific mRNAs by base-pairing with partially or fully complementary sequences in target mRNAs to modulate diverse biological and cellular processes. (3,4) Recent studies have suggested that miRNAs play crucial roles in osteoblast differentiation. (5) miR-133 and miR-135 impeded osteoblast differentiation by inhibiting the runt-related transcription factor 2 (Runx2) and SMAD family member 5 (Smad5) pathways that synergistically contributed to bone formation.…”