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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Santa Cruz Electronic Theses and Dissertations (2020)

Pre-mRNA splicing is a highly regulated step during gene expression and has been shown to be commonly altered across cancers. The basis for splicing alterations and the functional importance of cancer-associated spliced products remain largely unexplored. The scope of this work aims to better understand the basis for cancer-associated splicing alterations and their functional importance.

We first focus on establishing the genetic basis for cancer-associated splicing alterations. As part of the Pan Cancer Analysis of Whole Genomes (PCAWG) consortium, we demonstrate the impact of non-coding intronic mutations by using matched whole-genome and RNA-sequencing data across 1,209 primary tumor samples spanning 27 cancer types. We identify intronic sites beyond canonical acceptor and donor dinucleotides that are sensitive to mutations, including the branchpoint consensus sequences, which is typically missed in exome sequencing based tumor genotyping. We identify tumor suppressor genes and oncogenes with intronic mutations associated with substantial changes in splicing, and identify previously described alterations in the oncogene EZH2, as well as uncharacterized changes in oncogenes MET and HRAS. Altogether, this work provides the first estimates of the extent to which intronic mutations missed by exome-based genotyping contribute to splicing changes in cancer.

The second half of my work reveals the fate and function of spliced products associated with lung adenocarcinoma mutations in the splicing factor U2AF1. We conduct high-throughput long-read cDNA sequencing in isogenic human bronchial epithelial cells with and without U2AF1 S34F mutation. We demonstrate the utility of our long-read approach for transcriptome studies by identifying 49,366 novel isoforms exclusive to our approach. We show that our long-read data is robust for capturing mutant U2AF1-associated transcriptome alterations by comparing event-level alternative splicing changes with a short-read approach. We identify isoform-level expression changes in 198 isoforms, including a novel lncRNA, and immune-related genes. Last, we hypothesize a mechanism by which U2AF1 S34F alters translational control of genes through modulating isoform diversity.

    Cover page: Investigating cancer-associated pre-mRNA splicing alterations using short and long-read sequencing technologies
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2017)
    In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.
      Cover page: RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation
      • Article
      • Peer Reviewed
      UC Santa Cruz Previously Published Works (2023)
      U2AF1 is one of the most recurrently mutated splicing factors in lung adenocarcinoma and has been shown to cause transcriptome-wide pre-mRNA splicing alterations; however, the full-length altered mRNA isoforms associated with the mutation are largely unknown. To better understand the impact U2AF1 has on full-length isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without a U2AF1 S34F mutation. We identified 49,366 multi-exon transcript isoforms, more than half of which did not match GENCODE or short-read-assembled isoforms. We found 198 transcript isoforms with significant expression and usage changes relative to WT, only 68% of which were assembled by short reads. Expression of isoforms from immune-related genes is largely down-regulated in mutant cells and without observed splicing changes. Finally, we reveal that isoforms likely targeted by nonsense-mediated decay are down-regulated in U2AF1 S34F cells, suggesting that isoform changes may alter the translational output of those affected genes. Altogether, our work provides a resource of full-length isoforms associated with U2AF1 S34F in lung cells.
        Cover page: Full-length transcript alterations in human bronchial epithelial cells with U2AF1 S34F mutations
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2020)
        While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.
          Cover page: Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2019)
          Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.
            Cover page: Prp8 impacts cryptic but not alternative splicing frequency
            • Article
            • Peer Reviewed
            UC Santa Cruz Previously Published Works (2019)
            High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.
              Cover page: Nanopore native RNA sequencing of a human poly(A) transcriptome
              • Article
              • Peer Reviewed
              UC Santa Cruz Previously Published Works (2020)
              An amendment to this paper has been published and can be accessed via a link at the top of the paper.
                Cover page: Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.
                • Article
                • Peer Reviewed
                UC Santa Cruz Previously Published Works (2020)
                Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.
                  Cover page: Genomic basis for RNA alterations in cancer.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2020)
                  Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1-3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10-18.
                    Cover page: Pan-cancer analysis of whole genomes
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