The translational landscape of fission-yeast meiosis and sporulation.

Sexual development in Schizosaccharomyces pombe culminates in meiosis and sporulation. We used ribosome profiling to investigate the translational landscape of this process. We show that the translation efficiency of hundreds of genes is regulated in complex patterns, often correlating with changes in RNA levels. Ribosome-protected fragments show a three-nucleotide periodicity that identifies translated sequences and their reading frame. Using this property, we identified 46 new translated genes and found that 24% of noncoding RNAs are actively translated. We also detected 19 nested antisense genes, in which both DNA strands encode translated mRNAs. Finally, we identified 1,735 translated upstream open reading frames (ORFs) in leader sequences. In S. pombe, in contrast with Saccharomyces cerevisiae, sexual development is not accompanied by large increases in upstream ORF use, thus suggesting that this is an organism-specific adaptation, not a general feature of developmental processes.This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) research grant to Juan Mata (BB/J007153/1).This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/nsmb/journal/v21/n7/full/nsmb.2843.html

Structural analysis of protein-facilitated cooperative folding in the bI3 RNP

Most large RNAs require protein facilitators to achieve their active structures. Despite the importance of RNA structure for understanding the functions of associated proteins, initial and intermediate structures of the RNA are often assumed or not fully characterized. This gap in structural knowledge has lead to an over-simplification of the roles of many proteins in ribonucleoprotein complexes. Protein facilitators are currently categorized into two broad classes, cofactors and chaperones. Cofactors bind tightly an RNA to stabilize tertiary structures, while chaperones interact transiently with an RNA to facilitate acquisition of the most stable secondary structure. In this research, I comprehensively interrogate the structure of an RNA throughout the stages of protein-facilitated folding. In principle, the yeast bI3 group I intron has to potential to fold into the active secondary structure conserved among group I introns. The bI3 RNA requires two proteins (the bI3 maturase and Mrs1) for splicing. These proteins seem to have characteristics of cofactors and were not expected to affect the secondary structure. In contrast, by developing a high-throughput SHAPE experiment, I find that approximately half the bI3 RNA is not in the catalytically active secondary structure prior to protein binding. I develop and test a structural model for the misfolded RNA using SHAPE analysis of point mutations. I find that three conserved elements form stable, extensively mispaired, non-native structures. Solvent accessibility experiments show that these non-native structures are incapable of forming native group I intron tertiary interactions. I next demonstrate that binding by either the Mrs1 or maturase protein alone promotes the formation of distinct tertiary structures. However, neither individual protein binds to or affects the non-native secondary structures in the misfolded RNA. Strikingly, simultaneous binding of both proteins enables the RNA to achieve its active conformation. These results highlight a large-scale cooperative folding process between the bI3 RNA and the Mrs1 and maturase proteins in which the final RNA structure is drastically different than the sum of individual protein-bound states. The bI3 RNP thus represents a new category of ribonucleoprotein assembly mechanism in which binding by multiple proteins drives non-hierarchical RNA folding

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

The Mrs1 Splicing Factor Binds the bI3 Group I Intron at Each of Two Tetraloop-Receptor Motifs

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines

Widespread cotranslational formation of protein complexes.

Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly). We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA-binding domains, we found that ∼38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication. Both proteins interact with Cdc2p and are key cell cycle regulators. We obtained analogous results with proteins with different structures and cellular functions (kinesins, protein kinases, transcription factors, proteasome components, etc.). We showed that copurification of a bait protein and of specific mRNAs was dependent on the presence of the proteins encoded by the interacting mRNAs and on polysomal integrity. These results indicate that these observed associations reflect the cotranslational interaction between the bait and the nascent proteins encoded by the interacting mRNAs. Therefore, we show that the cotranslational formation of protein-protein interactions is a widespread phenomenon

Nonhierarchical Ribonucleoprotein Assembly Suggests a Strain-Propagation Model for Protein-Facilitated RNA Folding

Proteins play diverse and critical roles in cellular ribonucleoproteins (RNPs) including promoting formation of and stabilizing active RNA conformations. Yet, the conformational changes required to convert large RNAs into an active RNPs have proven difficult to characterize fully. Here we use high-resolution approaches to monitor both local nucleotide flexibility and solvent accessibility for nearly all nucleotides in the bI3 group I intron RNP in four assembly states: the free RNA, maturase-bound RNA, Mrs1-bound RNA, and the complete six-component holocomplex. The free RNA is misfolded relative to the secondary structure required for splicing. The maturase and Mrs1 proteins each stabilized long-range tertiary interactions but neither protein alone induced folding into the functional secondary structure. In contrast, simultaneous binding by both proteins results in large secondary structure rearrangements in the RNA and yielded the catalytically active group I intron structure. Secondary and tertiary folding of the RNA component of the bI3 RNP are thus not independent: RNA folding is strongly non-hierarchical. These results emphasize that protein-mediated stabilization of RNA tertiary interactions functions to pull the secondary structure into an energetically disfavored, but functional, conformation and emphasize a new role for facilitator proteins in RNP assembly

SHAPE Analysis of Long-Range Interactions Reveals Extensive and Thermodynamically Preferred Misfolding in a Fragile Group I Intron RNA †

Most functional RNAs require proteins to facilitate formation of their active structures. In the case of the yeast bI3 group I intron, splicing requires binding by two proteins, the intron-encoded bI3 maturase and the nuclear encoded Mrs1. Here, we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry coupled with analysis of point mutants to map long-range interactions in this RNA. This analysis reveals two critical features of the free RNA state. First, the catalytic intron is separated from the flanking exons via a stable anchoring helix. This anchoring helix creates an autonomous structural domain for the intron and functions to prevent misfolding with the flanking exons. Second, the thermodynamically most stable structure for the free RNA is not consistent with the catalytically active conformation as phylogenetically conserved elements form stable, non-native structures. These results highlight a fragile bI3 RNA for which binding by protein cofactors functions to promote extensive secondary structure rearrangements that are an obligatory prerequisite for forming the catalytically active tertiary structure

Global coordination of transcriptional control and mRNA decay during cellular differentiation

We have systematically identified the targets of the Schizosaccharomyces pombe RNA-binding protein Meu5p, which is transiently induced during cellular differentiation. Meu5p-bound transcripts (>80) are expressed at low levels and have shorter half-lives in meu5 mutants, suggesting that Meu5p binding stabilizes its RNA targets.Most Meu5p targets are induced during differentiation by the activity of the Mei4p transcription factor. However, although most Mei4p targets display a sharp peak of expression, Meu5p targets are expressed for a longer period. In the absence of Meu5p, all Mei4p targets are expressed with similar kinetics (similar to non-Meu5p targets). Therefore, Meu5p determines the temporal profile of its targets.As the meu5 gene is itself a target of the transcription factor Mei4p, the RNA-binding protein Meu5p and their shared targets form a feed-forward loop (FFL), a network motif that is common in transcriptional networks.Our data highlight the importance of considering both transcriptional and posttranscriptional controls to understand dynamic changes in RNA levels, and provide insight into the structure of the regulatory networks that integrate transcription and RNA decay

High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly

RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and nonadditive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes

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-