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Open Access Publications from the University of California

Scholarly Works (5 results)

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

Eukaryotic gene expression is an essential process for proper cell differentiation, development, and the cell’s response to environmental signals. Pre-mRNA splicing is an important part of the eukaryotic gene expression program. Splicing contributes to protein diversity, gene expression regulation, evolution, and genetic diseases. Understanding the intricacies of pre-mRNA splicing is important for understanding gene expression and for the rational design and development of new therapies for genetic diseases.The regulation of pre-mRNA splicing is a highly combinatorial process that relies on many cis- and trans-acting elements. Some of these elements include splice site strength and the intron-exon architecture. It is proposed that spliceosome assembly can either occur across the intron (referred to as intron definition) and across the exon (referred to as exon definition). Selecting between these modes of spliceosome assembly is thought to be dictated by intron architecture. Other studies have demonstrated that the proximity between the 5' splice site and its intronic 3' splice site plays a critical role in splice site selection. In chapter 2 we conducted a genome-wide computational analyses to evaluate the proximity rules in the context of intron and exon definition. Our computational studies were complemented using designer mini-genes in cell transfection assays to evaluate the impact of splice site proximity in alternative splicing. The ability to regulate gene expression allows the cell to adjust to its ever-changing needs and external cues. Aberrant regulation of gene expression is linked to diseases such as cancer. One major contributor to modulate gene expression is through the regulation of mRNA stability. A change in mRNA stability can lead to differing protein expression levels while alternative splicing primarily promotes protein diversity. The work described in Chapter 3 outlines what gene features impact mRNA stability and how alternative splicing can influence mRNA stability. Data was generated by conducting a 24-hour 4sU pulse-chase RNA-seq experiment. Our computational analyses allowed us to explore the relationship between mRNA stability and gene and/or exon length. In addition, we established a pipeline to derive exon and mRNA isoform half-lives to investigate the influence of alternative splicing on mRNA stability. Cancer cells have been known to have unique metabolic needs for proliferation. One such need is the cancer cell’s metabolic addiction to methionine, referred to as the “Hoffman effect.” While the Hoffman effect has been observed in a wide array of cancer cells, the mechanisms by which it arises, and controls tumorigenesis are not fully understood. In chapter 4, gene expression analyses of methionine-dependent and independent cell lines reveal that splicing dysregulation is linked to methionine dependence. In particular, proper methylation of a general spliceosomal component is implicated as link between changes in splicing fidelity and the accessibility of exogenous methionine in cancer cells.

    Cover page: The Impact of Pre-mRNA Splice Site Selection on mRNA Stability and Splicing Fidelity in Methionine-Dependent Cancer Cells
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2022)
    Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.
      Cover page: Splice site proximity influences alternative exon definition
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2017)
      Isolation of newly transcribed RNA is an invaluable approach that can be used to study the dynamic life of RNA in cellulo. Traditional methods of whole-cell RNA extraction limit subsequent gene expression analyses to the steady-state levels of RNA abundance, which often masks changes in RNA synthesis and processing. This chapter describes a methodology with low cytotoxicity that permits the labeling and isolation of nascent pre-mRNA in cell culture. The resulting isolate is suitable for use in a series of downstream applications aimed at studying changes in RNA synthesis, processing, or stability.
        Cover page: Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2015)
        The MED-1,2 GATA factors contribute to specification of E, the progenitor of the Caenorhabditis elegans endoderm, through the genes end-1 and end-3, and in parallel with the maternal factors SKN-1, POP-1 and PAL-1. END-1,3 activate elt-2 and elt-7 to initiate a program of intestinal development, which is maintained by positive autoregulation. Here, we advance the understanding of MED-1,2 in E specification. We find that expression of end-1 and end-3 is greatly reduced in med-1,2(-) embryos. We generated strains in which MED sites have been mutated in end-1 and end-3. Without MED input, gut specification relies primarily on POP-1 and PAL-1. 25% of embryos fail to make intestine, while those that do display abnormal numbers of gut cells due to a delayed and stochastic acquisition of intestine fate. Surviving adults exhibit phenotypes consistent with a primary defect in the intestine. Our results establish that MED-1,2 provide robustness to endoderm specification through end-1 and end-3, and reveal that gut differentiation may be more directly linked to specification than previously appreciated. The results argue against an "all-or-none" description of cell specification, and suggest that activation of tissue-specific master regulators, even when expression of these is maintained by positive autoregulation, does not guarantee proper function of differentiated cells.
          Cover page: MED GATA factors promote robust development of the C. elegans endoderm
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition. We scrutinized Sdic in 20 geographically diverse populations using reference-quality genome assemblies, read-depth methodologies, and qPCR, finding that ∼90% of the individuals harbor 3-7 copies as well as evidence of population differentiation. In strains with reliable gene annotations, copy number variation (CNV) and differential transposable element insertions distinguish one structurally distinct version of the Sdic region per strain. All 31 annotated copies featured protein-coding potential and, based on the protein variant encoded, were categorized into 13 paratypes differing in their 3' ends, with 3-5 paratypes coexisting in any strain examined. Despite widespread gene conversion, the only copy present in all strains has functionally diverged at both coding and regulatory levels under positive selection. Contrary to artificial tandem duplications of the Sdic region that resulted in increased male expression, CNV in cosmopolitan strains did not correlate with expression levels, likely as a result of differential genome modifier composition. Duplicating the region did not enhance sperm competitiveness, suggesting a fitness cost at high expression levels or a plateau effect. Beyond facilitating a minimally optimal expression level, Sdic CNV acts as a catalyst of protein and regulatory diversity, showcasing a possible evolutionary path recently formed tandem multigene families can follow toward long-term consolidation in eukaryotic genomes.
            Cover page: Understanding the early evolutionary stages of a tandem D. melanogaster-specific gene family: a structural and functional population study
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