Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Synonymous mutations in representative yeast genes are mostly strongly non-neutral.

Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral1-5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar-albeit nonidentical-between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

Replication stress inhibits synthesis of histone mRNAs in yeast by removing Spt10p and Spt21p from the histone promoters.

Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometry for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In mammalian cells, replication stress triggers degradation of histone mRNAs, but it is unclear if this mechanism is conserved from other species. The aim of this study was to identify the histone mRNA decay pathway in the yeast Saccharomyces cerevisiae and determine the mechanism by which DNA replication stress represses histone mRNAs. Using reverse transcription-quantitative PCR and chromatin immunoprecipitation-quantitative PCR, we show here that histone mRNAs can be degraded by both 5' → 3' and 3' → 5' pathways; however, replication stress does not trigger decay of histone mRNA in yeast. Rather, replication stress inhibits transcription of histone genes by removing the histone gene-specific transcription factors Spt10p and Spt21p from histone promoters, leading to disassembly of the preinitiation complexes and eviction of RNA Pol II from histone genes by a mechanism facilitated by checkpoint kinase Rad53p and histone chaperone Asf1p. In contrast, replication stress does not remove SCB-binding factor transcription complex, another activator of histone genes, from the histone promoters, suggesting that Spt10p and Spt21p have unique roles in the transcriptional downregulation of histone genes during replication stress. Together, our data show that, unlike in mammalian cells, replication stress in yeast does not trigger decay of histone mRNAs but inhibits histone transcription.

Transcription coordinates histone amounts and genome content.

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.

Identification of microRNA-like RNAs from Trichoderma asperellum DQ-1 during its interaction with tomato roots using bioinformatic analysis and high-throughput sequencing.

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast.

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.

Zinc-dependent activation of the Pho8 alkaline phosphatase in Schizosaccharomyces pombe.

Genome-wide analyses have revealed that during metal ion starvation, many cells undergo programmed changes in their transcriptome or proteome that lower the levels of abundant metalloproteins, conserving metal ions for more critical functions. Here we investigated how changes in cellular zinc status affect the expression and activity of the zinc-requiring Pho8 alkaline phosphatase from fission yeast (Schizosaccharomyces pombe). In S. pombe, Pho8 is a membrane-tethered and processed glycoprotein that resides in the vacuole. Using alkaline phosphatase activity assays along with various biochemical analyses, we found that Pho8 is active when zinc is plentiful and inactive when zinc is limited. Although Pho8 activity depended on zinc, we also found that higher levels of pho8 mRNAs and Pho8 protein accumulate in zinc-deficient cells. To gain a better understanding of the inverse relationship between pho8 mRNA levels and Pho8 activity, we examined the effects of zinc on the stability and processing of the Pho8 protein. We show that Pho8 is processed regardless of zinc status and that mature Pho8 accumulates under all conditions. We also noted that alkaline phosphatase activity is rapidly restored when zinc is resupplied to cells, even in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that S. pombe cells maintain inactive pools of Pho8 proteins under low-zinc conditions and that these pools facilitate rapid restoration of Pho8 activity when zinc ions become available.

RNA polymerase II (RNAP II)-associated factors are recruited to tRNA loci, revealing that RNAP II- and RNAP III-mediated transcriptions overlap in yeast.

In yeast (Saccharomyces cerevisiae), the synthesis of tRNAs by RNA polymerase III (RNAP III) down-regulates the transcription of the nearby RNAP II-transcribed genes by a mechanism that is poorly understood. To clarify the basis of this tRNA gene-mediated (TGM) silencing, here, conducting a bioinformatics analysis of available ChIP-chip and ChIP-sequencing genomic data from yeast, we investigated whether the RNAP III transcriptional machinery can recruit protein factors required for RNAP II transcription. An analysis of 46 genome-wide protein-density profiles revealed that 12 factors normally implicated in RNAP II-mediated gene transcription are more enriched at tRNA than at mRNA loci. These 12 factors typically have RNA-binding properties, participate in the termination stage of the RNAP II transcription, and preferentially localize to the tRNA loci by a mechanism that apparently is based on the RNAP III transcription level. The factors included two kinases of RNAP II (Bur1 and Ctk1), a histone demethylase (Jhd2), and a mutated form of a nucleosome-remodeling factor (Spt6) that have never been reported to be recruited to tRNA loci. Moreover, we show that the expression levels of RNAP II-transcribed genes downstream of tRNA loci correlate with the distance from the tRNA gene by a mechanism that depends on their orientation. These results are consistent with the notion that pre-tRNAs recruit RNAP II-associated factors, thereby reducing the availability of these factors for RNAP II transcription and contributing, at least in part, to the TGM-silencing mechanism.

5' processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes.

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation.

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Distribution shapes govern the discovery of predictive models for gene regulation.

Despite substantial experimental and computational efforts, mechanistic modeling remains more predictive in engineering than in systems biology. The reason for this discrepancy is not fully understood. One might argue that the randomness and complexity of biological systems are the main barriers to predictive understanding, but these issues are not unique to biology. Instead, we hypothesize that the specific shapes of rare single-molecule event distributions produce substantial yet overlooked challenges for biological models. We demonstrate why modern statistical tools to disentangle complexity and stochasticity, which assume normally distributed fluctuations or enormous datasets, do not apply to the discrete, positive, and nonsymmetric distributions that characterize mRNA fluctuations in single cells. As an example, we integrate single-molecule measurements and advanced computational analyses to explore mitogen-activated protein kinase induction of multiple stress response genes. Through systematic analyses of different metrics to compare the same model to the same data, we elucidate why standard modeling approaches yield nonpredictive models for single-cell gene regulation. We further explain how advanced tools recover precise, reproducible, and predictive understanding of transcription regulation mechanisms, including gene activation, polymerase initiation, elongation, mRNA accumulation, spatial transport, and decay.

Nucleus-specific expression in the multinuclear mushroom-forming fungus Agaricus bisporus reveals different nuclear regulatory programs.

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation.

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.

Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism.

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms.

Nascent RNA signaling to yeast RNA Pol II during transcription elongation.

Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed "RAPs". RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.

Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast.

The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step. The RNA is then reverse transcribed into cDNA using random nine-mers and oligo dT primers . Quantitative PCR using SYBR green is then performed to quantify RNA levels. This protocol has been tested on several S. pombe genotypes and generates highly reproducible results.

Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis.

The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives." Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.

Poly(A) site choice and Pol2 CTD Serine-5 status govern lncRNA control of phosphate-responsive tgp1 gene expression in fission yeast.

Expression of fission yeast glycerophosphate transporter Tgp1 is repressed in phosphate-rich medium and induced during phosphate starvation. Repression is enforced by transcription of the nc-tgp1 locus upstream of tgp1 to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the nc-tgp1 promoter: a TATA box -30TATATATA-23 and a HomolD box -64CAGTCACA-57, mutations of which inactivate the nc-tgp1 promoter and de-repress the downstream tgp1 promoter under phosphate-replete conditions. The nc-tgp1 lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (1632TCGGACATTCAA1643), the transcription factor that drives tgp1 expression. Overlap between the lncRNA template and the tgp1 promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the nc-tgp1 locus, polyadenylated at position +508, well upstream of the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal abolishes de-repression of the downstream tgp1 promoter elicited by Pol2 CTD Ser5Ala phospho-site mutation. Ser5 mutation favors utilization of the short RNA poly(A) site, thereby diminishing transcription of the lncRNA that interferes with the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal attenuates induction of the tgp1 promoter during phosphate starvation. Polyadenylation site choice governed by CTD Ser5 status adds a new level of lncRNA control of gene expression and reveals a new feature of the fission yeast CTD code.

Regulation of tRNA synthesis by posttranslational modifications of RNA polymerase III subunits.

RNA polymerase III (RNAPIII) transcribes tRNA genes, 5S RNA as well as a number of other non-coding RNAs. Because transcription by RNAPIII is an energy-demanding process, its activity is tightly linked to the stress levels and nutrient status of the cell. Multiple signaling pathways control RNAPIII activity in response to environmental cues, but exactly how these pathways regulate RNAPIII is still poorly understood. One major target of these pathways is the transcriptional repressor Maf1, which inhibits RNAPIII activity under conditions that are detrimental to cell growth. However, recent studies have found that the cell can also directly regulate the RNAPIII machinery through phosphorylation and sumoylation of RNAPIII subunits. In this review we summarize post-translational modifications of RNAPIII subunits that mainly have been identified in large-scale proteomics studies, and we highlight several examples to discuss their relevance for regulation of RNAPIII.