Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

Requirement for cleavage factor IIm in the control of alternative polyadenylation in breast cancer cells.

Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' untranslated regions (3'-UTRs), we observed shifts from promoter proximal regions to the 3'-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.

A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export.

Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis.

Co-translational control of protein complex formation: a fundamental pathway of cellular organization?

Analyses of proteomes from a large number of organisms throughout the domains of life highlight the key role played by multiprotein complexes for the implementation of cellular function. While the occurrence of multiprotein assemblies is ubiquitous, the understanding of pathways that dictate the formation of quaternary structure remains enigmatic. Interestingly, there are now well-established examples of protein complexes that are assembled co-translationally in both prokaryotes and eukaryotes, and indications are that the phenomenon is widespread in cells. Here, we review complex assembly with an emphasis on co-translational pathways, which involve interactions of nascent chains with other nascent or mature partner proteins, respectively. In prokaryotes, such interactions are promoted by the polycistronic arrangement of mRNA and the associated co-translation of functionally related cell constituents in order to enhance otherwise diffusion-dependent processes. Beyond merely stochastic events, however, co-translational complex formation may be sensitive to subunit availability and allow for overall regulation of the assembly process. We speculate how co-translational pathways may constitute integral components of quality control systems to ensure the correct and complete formation of hundreds of heterogeneous assemblies in a single cell. Coupling of folding of intrinsically disordered domains with co-translational interaction of binding partners may furthermore enhance the efficiency and fidelity with which correct conformation is attained. Co-translational complex formation may constitute a fundamental pathway of cellular organization, with profound importance for health and disease.

Coordination of Cell Cycle Progression and Mitotic Spindle Assembly Involves Histone H3 Lysine 4 Methylation by Set1/COMPASS.

Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and clustering of transcript abundance time series in synchronized cells revealed a requirement for Set1 for proper cell-cycle-dependent gene expression and Δset1 cells displayed delayed entry into S phase. Disruption of G1/S regulation in Δmbp1 and Δswi4 transcription factor mutants duplicated both benomyl resistance and suppression of ipl1-2 as was observed with Δset1 Taken together our results support a role for H3K4 methylation in the coordination of cell-cycle progression and proper assembly of the mitotic spindle during mitosis.

The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination.

During meiosis, combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to the initiation of double-strand breaks (DSBs) dependent on Spo11 remains unknown. Here, we show that the tethering of a PHD-containing protein, Spp1 (a component of the COMPASS complex), to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, by interacting with H3K4me3 and Mer2, Spp1 promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals.

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural 'linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regulatory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

The P-loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in pre-mRNA 3' end formation.

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.

Coupled RNA polymerase II transcription and 3' end formation with yeast whole-cell extracts.

RNA polymerase II (RNAP II) transcription and pre-mRNA 3' end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3' end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 3' end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m7G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 3' end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 5'-3' exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation.

Cordycepin (3' deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3' hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3' end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3' end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

The role of the putative 3' end processing endonuclease Ysh1p in mRNA and snoRNA synthesis.

Pre-mRNA 3' end formation is tightly linked to upstream and downstream events of eukaryotic mRNA synthesis. The two-step reaction involves endonucleolytic cleavage of the primary transcript followed by poly(A) addition to the upstream cleavage product. To further characterize the putative 3' end processing endonuclease Ysh1p/Brr5p, we isolated and analyzed a number of new temperature- and cold-sensitive mutant alleles. We show that Ysh1p plays a crucial role in 3' end formation and in RNA polymerase II (RNAP II) transcription termination on mRNA genes. In addition, we observed a range of additional functional deficiencies in ysh1 mutant strains, which were partially allele-specific. Interestingly, snoRNA 3' end formation and RNAP II termination were defective on specific snoRNAs in the cold-sensitive ysh1-12 strain. Moreover, we observed the accumulation of several mRNAs including the NRD1 transcript in this mutant. We provide evidence that NRD1 autoregulation is associated with endonucleolytic cleavage and that this process may involve Ysh1p. In addition, the ysh1-12 strain displayed defects in RNA splicing indicating that a functional link may exist between intron removal and 3' end formation in yeast. These observations suggest that Ysh1p has multiple roles in RNA synthesis and processing.

Transcriptional ShortCUTs.

Recent work from Kuehner and Brow (2008) and Thiebaut et al. (2008) in Molecular Cell and Jenks et al. (2008) in Molecular and Cellular Biology reveals that regulated expression of central nucleotide synthesis pathway components directs start site-dependent RNA polymerase II termination.

Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae.

To identify genes involved in poly(A) metabolism, we screened the yeast gene deletion collection for growth defects in the presence of cordycepin (3'-deoxyadenosine), a precursor to the RNA chain terminating ATP analog cordycepin triphosphate. Deltapho80 and Deltapho85 strains, which have a constitutively active phosphate-response pathway, were identified as cordycepin hypersensitive. We show that inorganic polyphosphate (poly P) accumulated in these strains and that poly P is a potent inhibitor of poly(A) polymerase activity in vitro. Binding analyses of poly P and yeast Pap1p revealed an interaction with a k(D) in the low nanomolar range. Poly P also bound mammalian poly(A) polymerase, however, with a 10-fold higher k(D) compared to yeast Pap1p. Genetic tests with double mutants of Deltapho80 and other genes involved in phosphate homeostasis and poly P accumulation suggest that poly P contributed to cordycepin hypersensitivity. Synergistic inhibition of mRNA synthesis through poly P-mediated inhibition of Pap1p and through cordycepin-mediated RNA chain termination may thus account for hypersensitive growth of Deltapho80 and Deltapho85 strains in the presence of the chain terminator. Consistent with this, a mutation in the 3'-end formation component rna14 was synthetic lethal in combination with Deltapho80. Based on these observations, we suggest that binding of poly P to poly(A) polymerase negatively regulates its activity.

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation.

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation.

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3' end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed beta-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3' end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3' end formation of specific mRNAs and snoRNAs. Protein-protein interaction studies support a role for Swd2p in the assembly of 3' end formation complexes. Furthermore, histone 3 lysine 4 di-and tri-methylation were adversely affected and telomeres were shortened in swd2 mutants. Underaccumulation of the Set1p methyltransferase accounts for the observed loss of SET1C activity and suggests a requirement for Swd2p for the stability or assembly of this complex. We also provide evidence that the roles of Swd2p as component of CPF and SET1C are functionally independent. Taken together, our results establish a dual requirement for Swd2p in 3' end formation and histone tail modification.

Coupling between snoRNP assembly and 3' processing controls box C/D snoRNA biosynthesis in yeast.

RNA polymerase II transcribes genes encoding proteins and a large number of small stable RNAs. While pre-mRNA 3'-end formation requires a machinery ensuring tight coupling between cleavage and polyadenylation, small RNAs utilize polyadenylation-independent pathways. In yeast, specific factors required for snRNA and snoRNA 3'-end formation were characterized as components of the APT complex that is associated with the core complex of the cleavage/polyadenylation machinery (core-CPF). Other essential factors were identified as independent components: Nrd1p, Nab3p and Sen1p. Here we report that mutations in the conserved box D of snoRNAs and in the snoRNP-specific factor Nop1p interfere with transcription and 3'-end formation of box C/D snoRNAs. We demonstrate that Nop1p is associated with box C/D snoRNA genes and that it interacts with APT components. These data suggest a mechanism of quality control in which efficient transcription and 3'-end formation occur only when nascent snoRNAs are successfully assembled into functional particles.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation.

Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3'-end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3'-end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3'-end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3'-end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination.

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre-mRNA 3' end processing, binds to the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3' end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3' end processing activity of Pcf11p and a deficiency of Pcf11p in 3' end processing did not prevent CTD binding. Transcriptional run-on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.