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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2018)

The eukaryotic genome is a large and complex network of molecules that work together to create diversity across many different species. Pre-mRNA splicing is a vital step in the processing of RNA into functionally diverse proteins. Splicing has a vast history and understanding the combination of many different factors and elements is fundamental to studying gene expression.

Gene evolution and the evolutionary pressures that encode a set of exonic sequences maintain efficient pre-mRNA splicing and ultimately dictate the selection of amino acids that define a protein. Exonic sequences have been regulated in a way to demonstrate the co-existence of coding and splicing pressures. Through the design of an exon conservation database, evolutionary conservation patterns were identified that influenced the final sequence of an exon. This information led to important predictions about splicing patterns in human disease. The database allowed for the identification of essential architectural parameters of the human genome. In addition, analysis of nucleotide variations at the wobble position identified splice altering SNPs and how these SNPs influenced exon inclusion.

Regulation of alternatively spliced exons requires a coordinated effort by many cis and trans-acting factors. Understanding how these factors work together is important for the mechanism of alternative splicing regulation. SR proteins and hnRNPs have previously demonstrated a position-dependent method of regulation. It was shown that U1 snRNP, at activating or repressive conditions, displayed dynamic changes in its compositional integrity. This demonstrated that U1 snRNP integrity is therefore modulated by the presence of position-dependent interactions with splicing regulatory factors, further suggestive of U1 snRNP as a molecular gatekeeper for splicing initiation.

Polyadenylation is a fundamental step in the 3’ end processing of mRNA. Alternative polyadenylation is another contributor to genomic diversity. The coordinated efforts between splicing and polyadenylation have been demonstrated for terminal exons, however, understanding the influence these two processes have on upstream exons was unknown. Genome-wide analyses allowed for the identification of an important role that a polyadenylation factor (CstF64) has on alternative splicing. In addition, the coupling that was seen between alternative polyadenylation and alternative splicing was in fact limited to terminal exons.

    Cover page: Molecular Mechanisms and Conservation of Pre-mRNA Splicing
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2017)
    The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.
      Cover page: Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2020)
      Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.
        Cover page: Allosteric regulation of U1 snRNP by splicing regulatory proteins controls spliceosomal assembly
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2019)
        Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size-filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size-filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size-filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.
          Cover page: Exon size and sequence conservation improves identification of splice-altering nucleotides
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2019)
          Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size-filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size-filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size-filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.
            Cover page: Exon size and sequence conservation improves identification of splice-altering nucleotides
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.
              Cover page: Allosteric regulation of U1 snRNP by splicing regulatory proteins controls spliceosomal assembly
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2014)
              The in vitro splicing assay is a valuable technique that can be used to study the mechanism and machinery involved in the splicing process. The ability to investigate various aspects of splicing and alternative splicing appears to be endless due to the flexibility of this assay. Here, we describe the tools and techniques necessary to carry out an in vitro splicing assay. Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis.
                Cover page: In Vitro Assay of Pre-mRNA Splicing in Mammalian Nuclear ExtractCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2016)
                Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.
                  Cover page: Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2016)
                  Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.
                    Cover page: Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2016)
                    Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.
                      Cover page: Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing.
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