A R-loop sensing pathway mediates the relocation of transcribed genes to nuclear pore complexes.

Nuclear pore complexes (NPCs) have increasingly recognized interactions with the genome, as exemplified in yeast, where they bind transcribed or damaged chromatin. By combining genome-wide approaches with live imaging of model loci, we uncover a correlation between NPC association and the accumulation of R-loops, which are genotoxic structures formed through hybridization of nascent RNAs with their DNA templates. Manipulating hybrid formation demonstrates that R-loop accumulation per se, rather than transcription or R-loop-dependent damages, is the primary trigger for relocation to NPCs. Mechanistically, R-loop-dependent repositioning involves their recognition by the ssDNA-binding protein RPA, and SUMO-dependent interactions with NPC-associated factors. Preventing R-loop-dependent relocation leads to lethality in hybrid-accumulating conditions, while NPC tethering of a model hybrid-prone locus attenuates R-loop-dependent genetic instability. Remarkably, this relocation pathway involves molecular factors similar to those required for the association of stalled replication forks with NPCs, supporting the existence of convergent mechanisms for sensing transcriptional and genotoxic stresses.

A dual, catalytic role for the fission yeast Ccr4-Not complex in gene silencing and heterochromatin spreading.

Heterochromatic gene silencing relies on combinatorial control by specific histone modifications, the occurrence of transcription, and/or RNA degradation. Once nucleated, heterochromatin propagates within defined chromosomal regions and is maintained throughout cell divisions to warrant proper genome expression and integrity. In the fission yeast Schizosaccharomyces pombe, the Ccr4-Not complex partakes in gene silencing, but its relative contribution to distinct heterochromatin domains and its role in nucleation versus spreading have remained elusive. Here, we unveil major functions for Ccr4-Not in silencing and heterochromatin spreading at the mating type locus and subtelomeres. Mutations of the catalytic subunits Caf1 or Mot2, involved in RNA deadenylation and protein ubiquitinylation, respectively, result in impaired propagation of H3K9me3 and massive accumulation of nucleation-distal heterochromatic transcripts. Both silencing and spreading defects are suppressed upon disruption of the heterochromatin antagonizing factor Epe1. Overall, our results position the Ccr4-Not complex as a critical, dual regulator of heterochromatic gene silencing and spreading.

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery.

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is not well understood. Here we demonstrate that Schizosaccharomyces pombe Lem2, an NE protein, regulates nuclear-exosome-mediated RNA degradation. Lem2 deletion causes accumulation of RNA precursors and meiotic transcripts and de-localization of an engineered exosome substrate from the nuclear periphery. Lem2 does not directly bind RNA but instead interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment. This pathway acts largely independently of nuclear bodies where exosome factors assemble. Nutrient availability modulates Lem2 regulation of meiotic transcripts, implying that this pathway is environmentally responsive. Our work reveals that multiple spatially distinct degradation pathways exist. Among these, Lem2 coordinates RNA surveillance of meiotic transcripts and non-coding RNAs by recruiting exosome co-factors to the nuclear periphery.

Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation.

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.

Long Non-Coding RNAs in the Control of Gametogenesis: Lessons from Fission Yeast.

Long non-coding RNAs (lncRNAs) contribute to cell fate decisions by modulating genome expression and stability. In the fission yeast Schizosaccharomyces pombe, the transition from mitosis to meiosis results in a marked remodeling of gene expression profiles, which ultimately ensures gamete production and inheritance of genetic information to the offspring. This key developmental process involves a set of dedicated lncRNAs that shape cell cycle-dependent transcriptomes through a variety of mechanisms, including epigenetic modifications and the modulation of transcription, post-transcriptional and post-translational regulations, and that contribute to meiosis-specific chromosomal events. In this review, we summarize the biology of these lncRNAs, from their identification to mechanism of action, and discuss their regulatory role in the control of gametogenesis.

A scaffold lncRNA shapes the mitosis to meiosis switch.

Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs.

ERH proteins: connecting RNA processing to tumorigenesis?

With the development of -omics approaches, the scientific community is now submerged by a wealth of information that can be used to analyze various parameters: the degree of protein sequence conservation, protein 3D structures as well as RNA and protein expression levels in various benign and tumor tissues, during organism development or upon exposure to chemicals such as endocrine disrupters. However, if such information can be used to identify genes with potentially important biological function, additional studies are needed to deeply characterize their cellular function in model organisms. Here, we discuss the case of such a gene: ERH, encoding a highly conserved homodimeric protein found in unicellular eukaryotes, plants and metazoan, of yet unknown biological function, which might be linked to mRNA metabolism and that is emerging as important for cell migration and metastasis.

Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression.

Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression.

Ubiquitination-dependent control of sexual differentiation in fission yeast.

In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.

High-frequency promoter firing links THO complex function to heavy chromatin formation.

The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single-molecule fluorescence in situ hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of gene-nuclear pore association and mRNP release to the same high pace of transcription initiation.

Ers1 links HP1 to RNAi.

Pericentromeric heterochromatin formation is mediated by repressive histone H3 lysine 9 methylation (H3K9Me) and its recognition by HP1 proteins. Intriguingly, in many organisms, RNAi is coupled to this process through poorly understood mechanisms. In Schizosaccharomyces pombe, the H3-K9 methyltransferase Clr4 and the heterochromatin protein 1 (HP1) ortholog Swi6 are critical for RNAi, whereas RNAi stimulates H3K9Me. In addition to the endoribonuclease Dcr1, RNAi in S. pombe requires two interacting protein complexes, the RITS complex, which contains an Argonaute subunit, and the RDRC complex, which contains an RNA-dependent RNA polymerase subunit. We previously identified Ers1 (essential for RNAi-dependent silencing) as an orphan protein that genetically acts in the RNAi pathway. Using recombinant proteins, we show here that Ers1 directly and specifically interacts with HP1/Swi6. Two-hybrid assays indicate that Ers1 also directly interacts with several RNAi factors. Consistent with these interactions, Ers1 associates in vivo with the RITS complex, the RDRC complex, and Dcr1, and it promotes interactions between these factors. Ers1, like Swi6, is also required for RNAi complexes to associate with pericentromeric noncoding RNAs. Overexpression of Ers1 results in a dominant-negative phenotype that can be specifically suppressed by increasing levels of the RDRC subunit Hrr1 or of Dcr1, further supporting a functional role for Ers1 in promoting the assembly of the RNAi machinery. Through the interactions described here, Ers1 may promote RNAi by tethering the corresponding enzyme complexes to HP1-coated chromatin, thereby placing them in proximity to the nascent noncoding RNA substrate.

Implication of Ccr4-Not complex function in mRNA quality control in Saccharomyces cerevisiae.

Production of messenger ribonucleoprotein particles (mRNPs) is subjected to quality control (QC). In Saccharomyces cerevisiae, the RNA exosome and its cofactors are part of the nuclear QC machinery that removes, or stalls, aberrant molecules, thereby ensuring that only correctly formed mRNPs are exported to the cytoplasm. The Ccr4-Not complex, which constitutes the major S. cerevisiae cytoplasmic deadenylase, has recently been implied in nuclear exosome-related processes. Consistent with a possible nuclear function of the complex, the deletion or mutation of Ccr4-Not factors also elicits transcription phenotypes. Here we use genetic depletion of the Mft1p protein of the THO transcription/mRNP packaging complex as a model system to link the Ccr4-Not complex to nuclear mRNP QC. We reveal strong genetic interactions between alleles of the Ccr4-Not complex with both the exosomal RRP6 and MFT1 genes. Moreover, Rrp6p-dependent in vivo QC phenotypes of Δmft1 cells can be rescued by codeletion of several Ccr4-Not components. We discuss how the Ccr4-Not complex may connect with the mRNP QC pathway.

Control of cryptic transcription in eukaryotes.

Over the last few years, the development of large-scale technologies has radically modified our conception of genome-wide transcriptional control by unveiling an unexpected high complexity of the eukaryotic transcriptome. In organisms ranging from yeast to human, a considerable number of novel small RNA species have been discovered in regions that were previously thought to be incompatible with high levels of transcription. Intriguingly, these transcripts, which are rapidly targeted for degradation by the exosome, appear to be devoid of any coding potential and may be the consequence of unwanted transcription events. However, the notion that an important fraction of these RNAs represent by-products of regulatory transcription is progressively emerging. In this chapter, we discuss the recent advances made in our understanding of the shape of the eukaryotic transcriptome. We also focus on the molecular mechanisms that cells exploit to prevent cryptic transcripts from interfering with the expression of protein-coding genes. Finally, we summarize data obtained in different systems suggesting that such RNAs may play a critical role in the regulation of gene expression as well as the evolution of genomes.

The Cul4-Ddb1(Cdt)² ubiquitin ligase inhibits invasion of a boundary-associated antisilencing factor into heterochromatin.

Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1(Cdt)² ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1(Cdt)² complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.

Control of cryptic transcription in eukaryotes.

Over the last few years, the development of large-scale technologies has radically modified our conception of genome-wide transcriptional control by unveiling an unexpected high complexity of the eukaryotic transcriptome. In organisms ranging from yeast to human, a considerable number of novel small RNA species have been discovered in regions that were previously thought to be incompatible with high levels of transcription. Intriguingly, these transcripts, which are rapidly targeted for degradation by the exosome, appear to be devoid of any coding potential and may be the consequence of unwanted transcription events. However, the notion that an important fraction of these RNAs representby-products of regulatory transcription is progressively emerging. In this chapter, we discuss the recent advances made in our understanding of the shape of the eukaryotic transcriptome. We also focus on the molecular mechanisms that cells exploit to prevent cryptic transcripts from interfering with the expression of protein-coding genes. Finally, we summarize data obtained in different systems suggesting that such RNAs may play a critical role in the regulation of gene expression as well as the evolution of genomes.

THO/Sub2p functions to coordinate 3'-end processing with gene-nuclear pore association.

During transcription, proteins assemble sequentially with nascent RNA to generate a messenger ribonucleoprotein particle (mRNP). The THO complex and its associated Sub2p helicase are functionally implicated in both transcription and mRNP biogenesis but their precise function remains elusive. We show here that THO/Sub2p mutation leads to the accumulation of a stalled intermediate in mRNP biogenesis that contains nuclear pore components and polyadenylation factors in association with chromatin. Microarray analyses of genomic loci that are aberrantly docked to the nuclear pore in mutants allowed the identification of approximately 400 novel validated target genes that require THO /Sub2p for efficient expression. Our data strongly suggests that the THO complex/Sub2p function is required to coordinate events leading to the acquisition of export competence at a step that follows commitment to 3'-processing.

Nuclear quality control of RNA polymerase II ribonucleoproteins in yeast: tilting the balance to shape the transcriptome.

From the very first time they set foot in the nuclear landscape till the end of their life, RNAs are packaged in ribonucleoproteins (RNPs) and face numerous processing steps to achieve function. To avoid the catastrophic consequences of naturally occurring processing errors, cells employ numerous quality control strategies. Focusing on yeast as a model system, we will review here how nuclear mechanisms ensure the proper assembly and maturation of mRNPs for their release in the cytoplasm, and highlight how these mechanisms are exploited to shape the RNA polymerase II transcriptome.

Ers1, a rapidly diverging protein essential for RNA interference-dependent heterochromatic silencing in Schizosaccharomyces pombe.

Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAi-independent silencing at the mat3M or tel2R loci. ers1Delta cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys(9) methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.

mRNA journey to the cytoplasm: attire required.

In eukaryotes, copying the genetic information from a DNA template into RNA is not sufficient itself to confer functional competence to the DNA-encoded message. mRNAs have to be processed by enzymes and packaged with proteins within nuclei to generate mRNP (messenger ribonucleoprotein) particles, before these can be exported to the cytoplasm. Processing and packaging factors are believed to interact with the nascent mRNA co-transcriptionally, which protects the highly reactive RNA molecule from a presumably aggressive nuclear environment while providing early commitment to its functional fate. In this review, we will describe the factors that are believed to provide the appropriate 'dress code' to the mRNA and the mechanisms underlying the proofreading events that guarantee its quality, focusing on yeast as a model system.

Chapter 10. Estimating nuclear mRNA decay in Saccharomyces cerevisiae.

A significant proportion of RNA decay in eukaryotes takes place in the nucleus. In addition to targeting nongenic RNAs such as products of processing and spurious transcription events, nuclear RNA decay also removes aberrant transcripts from regular genes. Moreover, a small list of genes utilizes nuclear RNA turnover to regulate their expression levels. This chapter highlights concepts and techniques used in our laboratory to estimate nuclear mRNA degradation in Saccharomyces cerevisiae. This chapter focuses on the nuclear RNA decay route of the heat-inducible HSP104 transcript in strains harboring an inactive THO/Sub2p mRNA export complex. The described approaches are readily adoptable to the analysis of other cases of nuclear mRNA degradation.