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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 (2022)

RNA-Seq has brought forth significant discoveries concerning aberrations in RNA processing, implicating RNA variants in a variety of diseases. Here, I will discuss cancer-associated RNA variation accompanying either somatic mutations in a splicing factor or changes in RNA base editor abundance. While many splice variants have been examined at an event-level with short reads, identifying full-length isoform changes may better elucidate the functional consequences of these variants in cancer. Thus, we have employed long-read technology to obtain full-length transcript sequences, developing a computational workflow called FLAIR (Full-length Alternative Isoform analysis of RNA) to identify high-confidence transcripts, differential transcript usage, and haplotype-specific transcripts. We performed nanopore sequencing of chronic lymphocytic leukemia patient samples containing SF3B1 mutation. With FLAIR, we are able to find patterns of aberrant splicing and a decrease in unproductive retained introns associated with SF3B1 mutation. Additionally, we have applied FLAIR to direct RNA sequencing reads to facilitate the identification of longer poly(A) tail lengths associated with intron retentions. Finally, we have sequenced H1975 lung adenocarcinoma cells with knockdown of ADAR, an enzyme that mediates A-to-I editing. We further improved our workflow to identify key inosine-isoform associations with the goal of clarifying the prominence of ADAR in tumorigenesis. Ultimately, our work demonstrates the utility of nanopore sequencing for augmenting cancer and splicing research.

    Cover page: Alternative RNA Processing in Cancers
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2016)
    The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations.
      Cover page: A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus
      • Article
      • Peer Reviewed
      UC Santa Cruz Previously Published Works (2024)

      Background

      RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously.

      Results

      We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.

      Conclusions

      Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.
        Cover page: Detecting haplotype-specific transcript variation in long reads with FLAIR2
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2023)
        BACKGROUND: RNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, short read RNA-Seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employed long-read technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We have developed a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generated nanopore data with high sequence accuracy of H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We applied our workflow to identify key inosine-isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.
          Creative Commons 'BY' version 4.0 license
          • 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 (2022)
              Alternative splicing generates distinct mRNA variants and is essential for development, homeostasis, and renewal. Proteins of the serine/arginine (SR)-rich splicing factor family are major splicing regulators that are broadly required for organ development as well as cell and organism viability. However, how these proteins support adult organ function remains largely unknown. Here, we used the continuously growing mouse incisor as a model to dissect the functions of the prototypical SR family protein SRSF1 during tissue homeostasis and renewal. We identified an SRSF1-governed alternative splicing network that is specifically required for dental proliferation and survival of progenitors but dispensable for the viability of differentiated cells. We also observed a similar progenitor-specific role of SRSF1 in the small intestinal epithelium, indicating a conserved function of SRSF1 across adult epithelial tissues. Thus, our findings define a regulatory mechanism by which SRSF1 specifically controls progenitor-specific alternative splicing events to support adult tissue homeostasis and renewal.
                Cover page: SRSF1 governs progenitor-specific alternative splicing to maintain adult epithelial tissue homeostasis and renewal
                • 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.
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