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Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2023)

Global methods for assaying translation have greatly improved our understanding of the protein-coding capacity of the genome. In particular, it is now possible to perform genome-wide and condition-specific identification of translation initiation sites through modified ribosome profiling methods that selectively capture initiating ribosomes (TIS- profiling). Here we apply this approach to meiotic and mitotic timepoints in budding yeast and show the surprising diversity of protein products that can be revealed by such methods.Chapter 2 describes our use of TIS-profiling to globally annotate translation initiation sites in yeast, with a particular focus on alternative N-terminally extended protein isoforms, which initiate from near-cognate (non-AUG) start codons upstream of annotated AUG start codons. We identified 149 genes with an extended isoform and show that these isoforms are produced in concert with canonical isoforms and are translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. Despite our success in identifying extended protein isoforms, high-confidence identification of new coding regions that entirely overlap annotated coding regions – including those that encode truncated protein isoforms – remained challenging. As described in Chapter 3, we developed a sensitive and robust algorithm focused on identifying N-terminally truncated proteins genome-wide, identifying 388 truncated protein isoforms. We performed extensive experimental validation of these truncated proteins and defined two general classes. The first set lack large portions of the annotated protein sequence and tend to be produced from a truncated transcript. We show two such cases, Yap5truncation and Pus1truncation, to have condition-specific regulation and functions that appear distinct from their respective annotated isoforms. The second set of N-terminally truncated proteins lack only a small region of the annotated protein and are less likely to be regulated by an alternative transcript isoform. Many localize to different subcellular compartments than their annotated counterpart, representing a common strategy for achieving dual localization of otherwise functionally identical proteins. Together these findings support the adoption of less static views of gene identity and a broader framework for considering the translational capacity of the genome.

    Cover page: Non-Canonical Protein Isoforms Produce Diversity of Protein Function and Localization in Budding Yeast
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2021)
    Global methods for assaying translation have greatly improved our understanding of the protein-coding capacity of the genome. In particular, it is now possible to perform genome-wide and condition-specific identification of translation initiation sites through modified ribosome profiling methods that selectively capture initiating ribosomes. Here we discuss our recent study applying such an approach to meiotic and mitotic timepoints in the simple eukaryote, budding yeast, as an example of the surprising diversity of protein products-many of which are non-canonical-that can be revealed by such methods. We also highlight several key challenges in studying non-canonical protein isoforms that have precluded their prior systematic discovery. A growing body of work supports expanded use of empirical protein-coding region identification, which can help relieve some of the limitations and biases inherent to traditional genome annotation approaches. Our study also argues for the adoption of less static views of gene identity and a broader framework for considering the translational capacity of the genome.
      Cover page: Rules are made to be broken: a “simple” model organism reveals the complexity of gene regulation
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2018)
      Recent genomic analyses have revealed pervasive translation from formerly unrecognized short open reading frames (sORFs) during yeast meiosis. Despite their short length, which has caused these regions to be systematically overlooked by traditional gene annotation approaches, meiotic sORFs share many features with classical genes, implying the potential for similar types of cellular functions. We found that sORF expression accounts for approximately 10-20% of the cellular translation capacity in yeast during meiotic differentiation and occurs within well-defined time windows, suggesting the production of relatively abundant peptides with stage-specific meiotic roles from these regions. Here, we provide arguments supporting this hypothesis and discuss sORF similarities and differences, as a group, to traditional protein coding regions, as well as challenges in defining their specific functions.
        Cover page: Strategies and Challenges in Identifying Function for Thousands of sORF‐Encoded Peptides in Meiosis
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2018)
        Protein degradation is known to be a key component of expression regulation for individual genes, but its global impact on gene expression has been difficult to determine. We analyzed a parallel gene expression dataset of yeast meiotic differentiation, identifying instances of coordinated protein-level decreases to identify new cases of regulated meiotic protein degradation, including of ribosomes and targets of the meiosis-specific anaphase-promoting complex adaptor Ama1. Comparison of protein and translation measurements over time also revealed that, although meiotic cells are capable of synthesizing protein complex members at precisely matched levels, they typically do not. Instead, the members of most protein complexes are synthesized imprecisely, but their protein levels are matched, indicating that wild-type eukaryotic cells routinely use post-translational adjustment of protein complex partner levels to achieve proper stoichiometry. Outlier cases, in which specific complex components show divergent protein-level trends, suggest timed regulation of these complexes.
          Cover page: Precise Post-translational Tuning Occurs for Most Protein Complex Components during Meiosis
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          Cellular stress responses often require transcription-based activation of gene expression to promote cellular adaptation. Whether general mechanisms exist for stress-responsive gene downregulation is less clear. A recently defined mechanism enables both up- and downregulation of protein levels for distinct gene sets by the same transcription factor via coordinated induction of canonical mRNAs and long undecoded transcript isoforms (LUTIs). We analyzed parallel gene expression datasets to determine whether this mechanism contributes to the conserved Hac1-driven branch of the unfolded protein response (UPRER), indeed observing Hac1-dependent protein downregulation accompanying the upregulation of ER-related proteins that typifies UPRER activation. Proteins downregulated by Hac1-driven LUTIs include those with electron transport chain (ETC) function. Abrogated ETC function improves the fitness of UPRER-activated cells, suggesting functional importance to this regulation. We conclude that the UPRER drives large-scale proteome remodeling, including coordinated up- and downregulation of distinct protein classes, which is partly mediated by Hac1-induced LUTIs.
            Cover page: Global Proteome Remodeling during ER Stress Involves Hac1-Driven Expression of Long Undecoded Transcript Isoforms
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2020)
            Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.
              Cover page: Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast
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