Michael J. Law

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.

Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction

The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem–loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.

Oxidative-stress-induced nuclear to cytoplasmic relocalization is required for Not4-dependent cyclin C destruction

The yeast cyclin-C–Cdk8p kinase complex represses the transcription of a subset of genes involved in the stress response. To relieve this repression, cyclin C is destroyed in cells exposed to H2O2 by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H2O2-induced cyclin C destruction. Not4p is required for H2O2-induced cyclin C destruction in vivo and polyubiquitylates cyclin C in vitro by utilizing Lys48, a ubiquitin linkage associated with directing substrates to the 26S proteasome. Before its degradation, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm. This translocation requires both the cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for cyclin C destruction. In addition, blocking cytoplasmic translocation slows the mRNA induction kinetics of two stress response genes repressed by cyclin C. Finally, a cyclin C derivative restricted to the cytoplasm is still subject to Not4p-dependent destruction, indicating that the degradation signal does not occur in the nucleus. These results identify a stress-induced proteolytic pathway regulating cyclin C that requires nuclear to cytoplasmic relocalization and Not4p-mediated ubiquitylation.

The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development

It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes.

Acetylation of the transcriptional repressor Ume6p allows efficient promoter release and timely induction of the meiotic transient transcription program in yeast.

ABSTRACT Differentiation programs require strict spatial and temporal control of gene transcription. Genes expressed during meiotic development in Saccharomyces cerevisiae display transient induction and repression. Early meiotic gene (EMG) repression during mitosis is achieved by recruiting both histone deacetylase and chromatin remodeling complexes to their promoters by the zinc cluster DNA binding protein Ume6p. Ume6p repression is relieved by ubiquitin-mediated destruction that is stimulated by Gcn5p-induced acetylation. In this report, we demonstrate that Gcn5p acetylation of separate lysines within the zinc cluster domain negatively impacts Ume6p DNA binding. Mimicking lysine acetylation using glutamine substitution mutations decreased Ume6p binding efficiency and resulted in partial derepression of Ume6p-regulated genes. Consistent with this result, molecular modeling predicted that these lysine side chains are adjacent to the DNA phosphate backbone, suggesting that acetylation inhibits Ume6p binding by electrostatic repulsion. Preventing acetylation did not impact final EMG induction levels during meiosis. However, a delay in EMG induction was observed, which became more severe in later expression classes, ultimately resulting in delayed and reduced execution of the meiotic nuclear divisions. These results indicate that Ume6p acetylation ensures the proper timing of the transient transcription program during meiotic development.

Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p

Acetyltransferases induce transcription by enhancing the activity of transcriptional activators and opening chromatin domains. A third avenue is described by which gene activation is accomplished by acetylation through the targeted destruction of the Ume6p repressor.

Fine-Tuning of Histone H3 Lys4 Methylation During Pseudohyphal Differentiation by the CDK Submodule of RNA Polymerase II

Transcriptional regulation is dependent upon the interactions between the RNA pol II holoenzyme complex and chromatin. RNA pol II is part of a highly conserved multiprotein complex that includes the core mediator and CDK8 subcomplex. In Saccharomyces cerevisiae, the CDK8 subcomplex, composed of Ssn2p, Ssn3p, Ssn8p, and Srb8p, is thought to play important roles in mediating transcriptional control of stress-responsive genes. Also central to transcriptional control are histone post-translational modifications. Lysine methylation, dynamically balanced by lysine methyltransferases and demethylases, has been intensively studied, uncovering significant functions in transcriptional control. A key question remains in understanding how these enzymes are targeted during stress response. To determine the relationship between lysine methylation, the CDK8 complex, and transcriptional control, we performed phenotype analyses of yeast lacking known lysine methyltransferases or demethylases in isolation or in tandem with SSN8 deletions. We show that the RNA pol II CDK8 submodule components SSN8/SSN3 and the histone demethylase JHD2 are required to inhibit pseudohyphal growth—a differentiation pathway induced during nutrient limitation—under rich conditions. Yeast lacking both SSN8 and JHD2 constitutively express FLO11, a major regulator of pseudohyphal growth. Interestingly, deleting known FLO11 activators including FLO8, MSS11, MFG1, TEC1, SNF1, KSS1, and GCN4 results in a range of phenotypic suppression. Using chromatin immunoprecipitation, we found that SSN8 inhibits H3 Lys4 trimethylation independently of JHD2 at the FLO11 locus, suggesting that H3 Lys4 hypermethylation is locking FLO11 into a transcriptionally active state. These studies implicate the CDK8 subcomplex in fine-tuning H3 Lys4 methylation levels during pseudohyphal differentiation.

The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MATa/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

The conserved histone deacetylase Rpd3 and the DNA binding regulator Ume6 repress BOI1's meiotic transcript isoform during vegetative growth in Saccharomyces cerevisiae

BOI1 and BOI2 are paralogs important for the actin cytoskeleton and polar growth. BOI1 encodes a meiotic transcript isoform with an extended 5′‐untranslated region predicted to impair protein translation. It is, however, unknown how the isoform is repressed during mitosis, and if Boi1 is present during sporulation. By interpreting microarray data from MATa cells, MATa/α cells, a starving MATα/α control, and a meiosis‐impaired rrp6 mutant, we classified BOI1s extended isoform as early meiosis‐specific. These results were confirmed by RNA‐Sequencing, and extended by a 5′‐RACE assay and Northern blotting, showing that meiotic cells induce the long isoform while the mitotic isoform remains detectable during meiosis. We provide evidence via motif predictions, an in vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6 histone deacetylase complex, which represses meiotic genes during mitosis, also prevents the induction of BOI1s 5′‐extended isoform in mitosis by direct binding of Ume6 to its URS1 target. Finally, we find that Boi1 protein levels decline when cells switch from fermentation to respiration and sporulation. The histone deacetylase Rpd3 is conserved, and eukaryotic genes frequently encode transcripts with variable 5′‐UTRs. Our findings are therefore relevant for regulatory mechanisms involved in the control of transcript isoforms in multi‐cellular organisms.