Abstract
RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing.
Highlights
RNA-dependent mechanisms play roles in gene silencing both at the transcriptional (TGS) and post-transcriptional (PTGS) levels in a broad range of organisms
We find that Red1 associates with an Mtr4-like helicase Mtl1 (SPAC 17H9.02), two zinc finger proteins, Red5 (SPBC337.12) and Ars2 (SPBC725.08), the RNA recognition motif (RRM)containing protein Rmn1 (SPBC902.04), and a serine- and proline-rich protein Iss10 (SPAC7D4.14c)
We find that the expression of meiotic genes is not affected by the absence of heterochromatic silencing machinery and that the H3K9 methylation at meiotic genes does not restrict RNA polymerase (Pol) II access, suggesting that the presence of this histone mark at meiotic gene loci is not associated with the assembly of functional heterochromatin
Summary
RNA-dependent mechanisms play roles in gene silencing both at the transcriptional (TGS) and post-transcriptional (PTGS) levels in a broad range of organisms. RITS recruits the Clr4–Rik1–Cul (CLRC) methyltransferase complex to catalyze the di- and trimethylation of histone H3 lysine 9 (H3K9) to initiate heterochromatin formation at specific chromosome regions (Moazed 2009). Another mechanism involving the Trf4–Air2–Mtr polyadenylation (TRAMP) complex broadly couples the transcription of cryptic unstable transcripts (CUTs) and other classes of aberrant RNA to degradation by the exosome, a multisubunit 3′ to 5′ exonuclease (LaCava et al 2005; Houseley et al 2006; Chlebowski et al 2013). Long noncoding RNAs (lncRNAs), akin to fission yeast centromeric RNAs, have recently emerged as important mediators of gene silencing in plant and animal cells, during development and differentiation (Lee 2012; Wierzbicki 2012)
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