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Open Access Publications from the University of California

Scholarly Works (17 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2014)

Splicing of eukaryotic pre-mRNAs often occurs co-transcriptionally; however, the components of the co-transcriptional environment that influence splicing remain incompletely defined. The work presented here discusses the effects of altering histone modifications, in particular H2B ubiquitination, and RNA Polymerase II (RNAPII) elongation rate on splicing in Saccharomyces cerevisiae.

To begin our exploration into the effect of the co-transcriptional environment on splicing, we interrogated the genetic, functional, and biochemical interactions of Npl3, an SR-like protein in S. cerevisiae that promotes co-transcriptional spliceosome assembly. Among many chromatin and transcription factors, we found that Npl3 genetically interacts with the histone H2B ubiquitination machinery and went on to show that strains lacking H2B ubiquitination have a mild splicing defect (Chapter 1). We subsequently capitalized on the observation that H2B ubiquitination-dependent processes are sensitive to high temperatures, and observed splicing defects upon inhibition of H2B ubiquitination or related modifications, H3K4 and H3K36 methylation, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analyses on intron-containing genes indicated that H2B ubiquitination stimulates recruitment of the early splicing factors onto nascent RNAs (Chapter 2).

It is challenging to nail down the precise mechanisms by which splicing responds to histone modifications - direct physical interactions between the modification and the spliceosome would offer locus- and modification-specific recruitment to promote splicing efficiency, but an additional variable, RNAPII elongation rate, had been shown to be a key player in metazoans. We therefore interrogated the effect on splicing of altering RNAPII elongation rate in S.cerevisiae, using an allelic series of point mutants that cause fast and slow elongation in vitro. Our splicing microarray experiments revealed that RNAPII speed and splicing efficiency are anti-correlated: at many genes, increased elongation rate caused decreased splicing efficiency, while decreased elongation rate increased splicing efficiency (Chapters 3 and 4).

Taken together, our data support the growing notion that histone modifications and RNAPII elongation rate exert gene-specific effects and potentially offer multiple locus-specific mechanisms for modulating splicing in a dynamic context.

    Cover page: Chromatin Marks and RNA Polymerase II Speed in pre-mRNA Splicing Control
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2019)
    As a central hub of cellular metabolism and signaling, the mitochondrion is a crucial organelle whose dysfunction can cause disease and whose activity is intimately connected to aging. We review how the mitochondrial network maintains proteomic integrity, how mitochondrial proteotoxic stress is communicated and resolved in the context of the entire cell, and how mitochondrial systems function in the context of organismal health and aging. A deeper understanding of how mitochondrial protein quality control mechanisms are coordinated across these distinct biological levels should help explain why these mechanisms fail with age and, ultimately, how routes to intervention might be attained.
      Cover page: Mitochondrial proteostasis in the context of cellular and organismal health and aging
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2014)
      Control of pre-mRNA splicing is a critical part of the eukaryotic gene expression process. Extensive evidence indicates that transcription and splicing are spatiotemporally coordinated and that most splicing events occur co-transcriptionally. A kinetic coupling model has been proposed in metazoans to describe how changing RNA Polymerase II (RNAPII) elongation rate can impact which alternative splice sites are used. In Saccharomyces cerevisiae, in which most spliced genes have only a single intron and splice sites adhere to a strong consensus sequence, we recently observed that splicing efficiency was sensitive to mutations in RNAPII that increase or decrease its elongation rate. Our data revealed that RNAPII speed and splicing efficiency are generally anti-correlated: at many genes, increased elongation rate caused decreased splicing efficiency, while decreased elongation rate increased splicing efficiency. An improved splicing phenotype was also observed upon deletion of SUB1, a condition in which elongation rate is slowed. We discuss these data in the context of a growing field and expand the kinetic coupling model to apply to both alternative splicing and splicing efficiency.
        Cover page: Adventures in time and space: splicing efficiency and RNA polymerase II elongation rate.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2014)
        We have achieved a residue-level resolution of genetic interaction mapping - a technique that measures how the function of one gene is affected by the alteration of a second gene - by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine.
          Cover page: Genetic interaction analysis of point mutations enables interrogation of gene function at a residue‐level resolution
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2014)

          Background information

          Commitment to splicing occurs co-transcriptionally, but a major unanswered question is the extent to which various modifications of chromatin, the template for transcription in vivo, contribute to the regulation of splicing.

          Results

          Here, we perform genome-wide analyses showing that inhibition of specific marks - H2B ubiquitylation, H3K4 methylation and H3K36 methylation - perturbs splicing in budding yeast, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analysis on intron-containing genes indicated that H2B ubiquitylation, but not Set1-, Set2- or Dot1-dependent H3 methylation, stimulates recruitment of the early splicing factors, namely U1 and U2 snRNPs, onto nascent RNAs.

          Conclusions

          These results suggest that histone modifications impact splicing of distinct subsets of genes using distinct pathways.
            Cover page: H2B ubiquitylation modulates spliceosome assembly and function in budding yeast
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2012)
            Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing.
              Cover page: The Yeast SR-Like Protein Npl3 Links Chromatin Modification to mRNA Processing
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2017)
              Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.
                Cover page: Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2019)
                Somatic cells can be reprogrammed into pluripotent stem cells using the Yamanaka transcription factors. Reprogramming requires both epigenetic landscape reshaping and global remodeling of cell identity, structure, basic metabolic processes, and organelle form and function. We hypothesize that variable regulation of the proteostasis network and its influence upon the protein-folding environment within cells and their organelles is responsible for the low efficiency and stochasticity of reprogramming. We find that the unfolded protein response of the endoplasmic reticulum (UPRER), the mitochondrial UPR, and the heat shock response, which ensure proteome quality during stress, are activated during reprogramming. The UPRER is particularly crucial, and its ectopic, transient activation, genetically or pharmacologically, enhances reprogramming. Last, stochastic activation of the UPRER predicts reprogramming efficiency in naïve cells. Thus, the low efficiency and stochasticity of cellular reprogramming are due partly to the inability to properly initiate the UPRER to remodel the ER and its proteome.
                  Cover page: Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2019)
                  Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPRER, which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPRER pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPRER activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1.
                    Cover page: The Hyaluronidase, TMEM2, Promotes ER Homeostasis and Longevity Independent of the UPRER
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2021)
                    The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPRMT). However, it is still unclear how UPRMT is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPRMT. We performed cross-species comparison of genetic requirements for survival under—and in response to—mitochondrial stress between karyotypically normal human stem cells and the nematode Caenorhabditis elegans. We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPRMT regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.
                      Cover page: Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization
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