Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast

We uncovered a novel role for the spliceosome in regulating mRNA expression levels that involves splicing coupled to RNA decay, which we refer to as spliceosome-mediated decay (SMD). Our transcriptome-wide studies identified numerous transcripts that are not known to have introns but are spliced by the spliceosome at canonical splice sites in Saccharomyces cerevisiae. Products of SMD are primarily degraded by the nuclear RNA surveillance machinery. We demonstrate that SMD can significantly down-regulate mRNA levels; splicing at canonical splice sites in the bromodomain factor 2 (BDF2) transcript reduced transcript levels roughly threefold by generating unstable products that are rapidly degraded by the nuclear surveillance machinery. Regulation of BDF2 mRNA levels by SMD requires Bdf1, a functionally redundant Bdf2 paralog that plays a role in recruiting the spliceosome to the BDF2 mRNA. Interestingly, mutating BDF2 5' splice site and branch point consensus sequences partially suppresses the bdf1Δ temperature-sensitive phenotype, suggesting that maintaining proper levels of Bdf2 via SMD is biologically important. We propose that the spliceosome can also repress protein-coding gene expression by promoting nuclear turnover of spliced RNA products and provide an insight for coordinated regulation of Bdf1 and Bdf2 levels in the cell

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Background TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Results Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Conclusions Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity

Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in S. cerevisiae

The small GTPase Ypt1p of the Rab family is required for docking of ER-derived transport vesicles with the Golgi prior to fusion. However, the identity of the Rab protein that mediates docking of Golgi-derived COPI vesicles with the ER in retrograde transport remains elusive. Here, we show that in yeast Ypt1p is essential for retrograde transport from the Golgi to the ER. Retrieval of gpalphaF-HDEL (glycolylated pro-alpha-factor with an HDEL tag at the C-terminus) was blocked in Deltaypt1/SLY1-20 membranes at the restrictive temperature in vitro. Moreover, Ypt1p and the ER-resident t-SNARE Ufe1p interact genetically and biochemically, indicating a role for Ypt1p in consumption of COPI vesicles at the ER. Ypt1p is also essential for the maintenance of the morphology and the protein composition of the Golgi. Interestingly, the concentrations of the Golgi enzymes Anp1p and Mnn1p, the cargo protein Emp47p and the v-SNARE Sec22p were all substantially reduced in Golgi from a Deltaypt1/SLY1-20 strain as compared with wild-type Golgi, while the concentration of Arf1p and of coatomer were mildly affected. Finally, COPI vesicles generated from Deltaypt1/SLY1-20 Golgi membranes in vitro were depleted of Emp47p and Sec22p. These data demonstrate that Ypt1p plays an essential role in retrograde transport from the Golgi to the ER

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA

AbstractTermination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination