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Open Access Publications from the University of California

Scholarly Works (16 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2022)

Single-nucleotide variants (SNVs), such as genetic variants and RNA editing sites, constitute the most prevalent type of sequence variations in the RNA. Genome-wide association studies (GWAS) and global analysis of RNA editing have revealed many genetic variants and RNA editing sites associated with human diseases and complex traits. Yet, the underlying mechanisms of such associations are still missing for most SNVs. In this dissertation, we studied both non-coding and coding SNVs, revealing their functional roles in regulating mRNA abundance and splicing.

Understanding the function of non-coding rare genetic variants remains a major challenge. To fill in this gap, we developed a massively parallel reporter assay, allowing functional testing of 3’ UTR variants regulating mRNA abundance in a high-throughput manner. We screened 14,575 rare 3’ UTR genetic variants and identified 5,437 functional ones leading to significant changes in mRNA abundance in at least one human cell line. Supported by TCGA expression outlier analysis and experimental studies, we observed that many rare 3’ UTR variants regulate mRNA abundance of cancer-relevant genes.

We next examined a specific set of RNA editing sites that are differentially edited between epithelial and mesenchymal tumors across 7 cancer types in The Cancer Genome Atlas (TCGA). Inspired by the correlations between editing levels and gene expression, we uncovered many 3’ UTR RNA editing sites regulating mRNA abundance. Further, we identified the RNA-binding protein ILF3 as a potential regulator of the editing-dependent gene expression change, especially for immune-relevant genes. We showed that multiple RNA editing sites mediate the ILF3 stabilizing effect on the transcripts encoding Protein Kinase R (PKR), a key player in immune response.

In addition to non-coding variants, we further characterized two RNA editing sites in the alternative exon of the gene podocalyxin-like (PODXL), a significant clinical indicator for tumor detection and prognosis. Supported by survival analysis in Kidney Renal Clear Cell Carcinoma (KIRC) patients, we discovered dual roles of RNA editing in promoting the loss of function of PODXL in cancer. We hypothesized that exonic RNA editing sites contribute to proteomic diversity through alternative splicing, a previously overlooked function of RNA editing.

    Cover page: Deciphering the Function of Single Nucleotide Variants in Post-transcriptional Gene Regulation
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)
    PODXL, a protein that is dysregulated in multiple cancers, plays an important role in promoting cancer metastasis. In this study, we report that RNA editing promotes the inclusion of a PODXL alternative exon. The resulting edited PODXL long isoform is more prone to protease digestion and has the strongest effects on reducing cell migration and cisplatin chemoresistance among the three PODXL isoforms (short, unedited long, and edited long isoforms). Importantly, the editing level of the PODXL recoding site and the inclusion level of the PODXL alternative exon are strongly associated with overall patient survival in Kidney Renal Clear Cell Carcinoma (KIRC). Supported by significant enrichment of exonic RNA editing sites in alternatively spliced exons, we hypothesize that exonic RNA editing sites may enhance proteomic diversity through alternative splicing, in addition to amino acid changes, a previously under-appreciated aspect of RNA editing function.
      Cover page: Multifaceted role of RNA editing in promoting loss-of-function of PODXL in cancer
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2023)
      Aptamers are single-stranded DNA or RNA sequences that can specifically bind with the target protein or molecule via specific secondary structures. Compared to antibody-drug conjugates (ADC), aptamer‒drug conjugate (ApDC) is also an efficient, targeted drug for cancer therapy with a smaller size, higher chemical stability, lower immunogenicity, faster tissue penetration, and facile engineering. Despite all these advantages, several key factors have delayed the clinical translation of ApDC, such as in vivo off-target effects and potential safety issues. In this review, we highlight the most recent progress in the development of ApDC and discuss solutions to the problems noted above.
        Cover page: Recent progress of aptamer‒drug conjugates in cancer therapy.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2015)
        A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.
          Cover page: A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Although long-read RNA-seq is increasingly applied to characterize full-length transcripts it can also enable detection of nucleotide variants, such as genetic mutations or RNA editing sites, which is significantly under-explored. Here, we present an in-depth study to detect and analyze RNA editing sites in long-read RNA-seq. Our new method, L-GIREMI, effectively handles sequencing errors and read biases. Applied to PacBio RNA-seq data, L-GIREMI affords a high accuracy in RNA editing identification. Additionally, our analysis uncovered novel insights about RNA editing occurrences in single molecules and double-stranded RNA structures. L-GIREMI provides a valuable means to study nucleotide variants in long-read RNA-seq.
            Cover page: L-GIREMI uncovers RNA editing sites in long-read RNA-seq
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2020)

            Background

            RNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance.

            Results

            Here, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response.

            Conclusions

            Our study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.
              Cover page: RNA editing in cancer impacts mRNA abundance in immune response pathways
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2022)
              The zebra finch (Taeniopygia guttata), a representative oscine songbird species, has been widely studied to investigate behavioral neuroscience, most notably the neurobiological basis of vocal learning, a rare trait shared in only a few animal groups including humans. In 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from the Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. We further validated 18 different novel proteins by using mass-spectrometry data generated from zebra finch caudal telencephalon tissue. Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.
                Cover page: Improved zebra finch brain transcriptome identifies novel proteins with sex differences
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2023)
                RNA editing, the endogenous modification of nucleic acids, is known to be altered in genes with important neurological function in schizophrenia (SCZ). However, the global profile and molecular functions of disease-associated RNA editing remain unclear. Here, we analyzed RNA editing in postmortem brains of four SCZ cohorts and uncovered a significant and reproducible trend of hypoediting in patients of European descent. We report a set of SCZ-associated editing sites via WGCNA analysis, shared across cohorts. Using massively parallel reporter assays and bioinformatic analyses, we observed that differential 3' untranslated region (3'UTR) editing sites affecting host gene expression were enriched for mitochondrial processes. Furthermore, we characterized the impact of two recoding sites in the mitofusin 1 (MFN1) gene and showed their functional relevance to mitochondrial fusion and cellular apoptosis. Our study reveals a global reduction of editing in SCZ and a compelling link between editing and mitochondrial function in the disease.
                  Cover page: Widespread RNA hypoediting in schizophrenia and its relevance to mitochondrial function
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2024)
                  Bile acids (BAs) affect the intestinal environment by ensuring barrier integrity, maintaining microbiota balance, regulating epithelium turnover, and modulating the immune system. As a master regulator of BA homeostasis, farnesoid X receptor (FXR) is severely compromised in patients with inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). At the front line, gut macrophages react to the microbiota and metabolites that breach the epithelium. We aim to study the role of the BA/FXR axis in macrophages. This study demonstrates that inflammation-induced epithelial abnormalities compromised FXR signaling and altered BAs' profile in a mouse CAC model. Further, gut macrophage-intrinsic FXR sensed aberrant BAs, leading to pro-inflammatory cytokines' secretion, which promoted intestinal stem cell proliferation. Mechanistically, activation of FXR ameliorated intestinal inflammation and inhibited colitis-associated tumor growth, by regulating gut macrophages' recruitment, polarization, and crosstalk with Th17 cells. However, deletion of FXR in bone marrow or gut macrophages escalated the intestinal inflammation. In summary, our study reveals a distinctive regulatory role of FXR in gut macrophages, suggesting its potential as a therapeutic target for addressing IBD and CAC.
                    Cover page: Farnesoid X receptor mediates macrophage-intrinsic responses to suppress colitis-induced colon cancer progression.Creative Commons 'BY-NC-ND' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2024)
                    Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3' UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.
                      Cover page: Massively parallel screen uncovers many rare 3′ UTR variants regulating mRNA abundance of cancer driver genes
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