The Proper Splicing of RNAi Factors Is Critical for Pericentric Heterochromatin Assembly in Fission Yeast

Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres

The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport

Cwf14 is required for pericentric heterochromatin assembly through the RNAi pathway.

<p>(A, E, F, and G) Serial dilution analyses to measure the expression of reporter genes and sensitivity to TBZ. (B and C) ChIP analyses of Pol II (Rpb1), H3K9me2, and Swi6 levels at pericentric <i>dh</i> repeats and <i>otr::ura4⁺</i>. Pol II ChIP was normalized to <i>act1</i> gene, and H3K9me2 and Swi6 ChIP was normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments. (D) Northern blot analyses of siRNAs derived from pericentric <i>dh</i> repeats.</p

Introducing cDNAs of RNAi factors rescues pericentric heterochromatin defects of <i>cwf14Δ</i> cells.

<p>(A) Serial dilution analyses to measure the expression of <i>otr::ura4⁺</i> and sensitivity to TBZ. (B) ChIP analyses of H3K9me2 and Swi6 levels at pericentric <i>dh</i> repeats and <i>otr::ura4⁺</i>, normalized to an <i>act1</i> promoter fragment. Error bars represent standard deviation of three experiments.</p

Cwf14 is required for the proper splicing of RNAi factors.

<p>(A) Area-proportional overlap of genes up-regulated in <i>cwf14Δ</i> and <i>dcr1Δ</i> (each compared to wild type) at p<0.05. (B) Ratios of exon-exon junction counts from RNA-seq for each junction in the indicated samples. A ratio of 1 indicates no difference. The bottom tail of <i>cwf14Δ</i>/WT, which is less pronounced in <i>dcr1Δ/</i>WT, indicates mis-splicing. (C) RNA-seq analysis shows mis-splicing of a number of RNAi factor introns in <i>cwf14Δ</i>. Ratios are exon-exon junction counts from RNA-seq for each junction in the indicated samples. (D) Top, diagram of RNAi genes with intron positions indicated. Bars represent PCR fragments used to analyze intron retention. Bottom, RT-PCR analyses of RNA with primers flanking introns.</p

Splicing factors regulate the proper splicing of shelterin component Tpz1.

<p>(A) RT-PCR analyses of RNA with primers flanking introns. (B, E) Serial dilution analyses to measure reporter gene expression. (C) ChIP analyses of H3K9me2 levels, normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments. (D, F) qRT-PCR analyses of transcripts derived from <i>tlh1</i>, normalized to <i>act1</i> gene. Wild type was set to 1. Error bars represent standard deviation of three experiments.</p

Cwf14 associates with the spliceosome.

<p>(A) Tagged versions of Cwf14 are functional as indicated by serial dilution analysis. (B) Imaging of cells expressing Cwf14-GFP, with DAPI to stain nucleus. Scale bar, 10 µm. (C) Western blot analysis of Cwf14-myc expression. (D) Mass spectrometry analyses of purified Cwf14-myc complex. Factors were binned into annotated subcomplexes.</p

Cwf14 is not required for CLRC activity.

<p>(A) Schematic diagram of the <i>ura4::3xgbs-ade6⁺</i> reporter. (B) Left, serial dilution analysis to measure reporter gene expression. Right, ChIP analysis of H3K9me2 levels at the reporter, normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments.</p

A genetic screen for nonessential genes required for pericentric heterochromatin silencing in fission yeast.

<p>(A) Schematic diagram of the <i>otr::ade6⁺-natMX6</i> reporter. (B) Workflow to introduce <i>otr::ade6⁺</i> into the deletion library. A control screen was performed in parallel. The selection of only <i>ade6-M210-hphMX6</i> progeny generates a uniform background for color development. White arrows denote a mutant that affects silencing, and yellows arrows denote a false positive that affects colony color independently of silencing. (C) List of mutants identified affecting pericentric silencing. Hit colonies were assigned color scores between 1 and 4, as indicated. (D) qRT-PCR analyses of transcripts derived from pericentric <i>dh</i> repeats, normalized to <i>act1</i>. Wild-type was set to 1. Error bars represent standard deviation of three experiments.</p