Systematic analysis of the role of RNA-binding proteins in the regulation of RNA stability.

mRNA half-lives are transcript-specific and vary over a range of more than 100-fold in eukaryotic cells. mRNA stabilities can be regulated by sequence-specific RNA-binding proteins (RBPs), which bind to regulatory sequence elements and modulate the interaction of the mRNA with the cellular RNA degradation machinery. However, it is unclear if this kind of regulation is sufficient to explain the large range of mRNA stabilities. To address this question, we examined the transcriptome of 74 Schizosaccharomyces pombe strains carrying deletions in non-essential genes encoding predicted RBPs (86% of all such genes). We identified 25 strains that displayed changes in the levels of between 4 and 104 mRNAs. The putative targets of these RBPs formed biologically coherent groups, defining regulons involved in cell separation, ribosome biogenesis, meiotic progression, stress responses and mitochondrial function. Moreover, mRNAs in these groups were enriched in specific sequence motifs in their coding sequences and untranslated regions, suggesting that they are coregulated at the posttranscriptional level. We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities. Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains. This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.This work was supported by a Biotechnology and Biological Sciences Research Council grant BB/J007153/1 to JM (http://www.bbsrc.ac.uk), a Masdar Institute fellowship to AH (http://www.masdar.ac.ae/) and a Herchel Smith Postdoctoral fellowship to CC (http://www.herchelsmith.cam.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final published version. It first appeared at http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004684

Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

BACKGROUND: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway. RESULTS: Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner. CONCLUSIONS: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.This work was supported by a Biotechnology and Biological Sciences Research Council grant BB/J007153/1 to JM (http://www.bbsrc.ac.uk), a Masdar Institute fellowship to AH (http://www.masdar.ac.ae/), a Herchel Smith Postdoctoral Fellowship to CC (http://www.herchelsmith.cam.ac.uk), and a Wellcome Trust Senior Investigator Award 095598/Z/11/Z to JB (http://www.wellcome.ac.uk). We thank M. A. Rodríguez-Gabriel, F. Ishikawa and T. Sugiyama for providing strains.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13072-015-0018-

Correction: Functional profiling of long intergenic non-coding RNAs in fission yeast

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse codinggene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins

Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance

Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast.

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance

Potential regulatory elements.

<p>Sequence motifs enriched in mRNAs affected by deletions in the specified genes. Motifs were identified in coding sequences or 3′-UTRs as indicated. The sequence logos show the conservation at each position measured in bits (overall height of the stack), and the height of the symbols within the stack is proportional to the relative abundance of the bases.</p

Changes in intron levels caused by RBP inactivation.

<p>(A) Transcriptome of <i>SPBC18H1.07</i> mutant cells. The y and x axes show expression levels in mutant and wild type cells, respectively. Each dot corresponds to a different gene. Introns overexpressed in the mutant are shown in red, and the coding sequences of the corresponding genes in blue. Note that introns of the affected genes, but not their exons, are affected. (B) Overlap between introns overexpressed in <i>SPBC18H10.07</i> and <i>SPAC30D11.14</i> mutants. The number in brackets corresponds to the expected overlap if randomly-generated lists of the corresponding sizes were used. The p value of the observed overlap is shown below the Venn diagram. (C) Transcriptome of <i>red1</i> mutants. Labelling as in A. Introns overexpressed in <i>red1</i> cells are shown in red, and the corresponding coding sequences in blue. Note that both introns and exons of the affected genes are frequently overexpressed. The arrows mark the position of the <i>rpl3002</i> exon and intron probes. (D) Overlap between introns overexpressed in <i>pab2</i> and <i>red1</i> mutants. Labelling as in B.</p

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals