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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

The content and expression of the eukaryotic transcriptome are tightly regulated by sequence elements in the RNA. Understandably, genetic variations in these essential sequences can alter many aspects of the transcriptome. However, to date, the rules underlying RNA regulation by sequence elements remain perplexing. In this dissertation, we provide new insights into the effects of genetic variants on alternative splicing and RNA abundance as well as the molecular mechanisms underlying these functional effects.

While our understanding of rare variants remains elusive, their complexity is further heightened by their occurrence in noncoding regions of the genome, such as the 3’ untranslated region (UTR). To address this challenge, we developed a massively parallel reporter assay, called MapUTR, to identify post-transcriptional genetic regulators of mRNA abundance. We tested 17,301 rare single nucleotide variants (SNVs), a significant portion of which demonstrated a functional role in mRNA abundance regulation through miRNA and/or RNA binding protein (RBP) interactions. Additionally, we assayed 11,929 cancer somatic mutations, allowing us to characterize the potential impact of noncoding mutations on carcinogenesis and prognosis.

Next, we investigated the global regulatory effects of exonic variants on alternative splicing using next-generation sequencing datasets generated by the Genotype-Tissue Expression (GTEx) project. Our analyses revealed >18,000 SNVs associated with >12,000 exons subject to a genetically modulated alternative splicing (GMAS) program. We found that both the genetic background and tissue environment contributed to a similar extent to the GMAS pattern, although the allelic imbalance varied largely between samples. We also developed a computational method to pinpoint functional splice-disrupting variants and further highlighted their relevance to phenotypic traits and diseases.

Finally, we leveraged long-read sequencing data from ENCODE to segregate alternatively spliced exons that are primarily directed by cis-regulatory elements or trans-acting factors, using a novel method – isoLASER. We identified 2,807 and 5,322 unique exonic parts from human and mouse tissues under cis-directed splicing regulation. We demonstrate that alternative splicing in general is governed by tissue-specific factors whereas cis-directed splicing is dominated by the genetic background. The cis-directed events overlapped repeat elements and showed significant enrichment in genes related to immune function.

    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2024)
    Recent studies have highlighted the essential role of RNA splicing, a key mechanism of alternative RNA processing, in establishing connections between genetic variations and disease. Genetic loci influencing RNA splicing variations show considerable influence on complex traits, possibly surpassing those affecting total gene expression. Dysregulated RNA splicing has emerged as a major potential contributor to neurological and psychiatric disorders, likely due to the exceptionally high prevalence of alternatively spliced genes in the human brain. Nevertheless, establishing direct associations between genetically altered splicing and complex traits has remained an enduring challenge. We introduce Spliced-Transcriptome-Wide Associations (SpliTWAS) to integrate alternative splicing information with genome-wide association studies to pinpoint genes linked to traits through exon splicing events. We applied SpliTWAS to two schizophrenia (SCZ) RNA-sequencing datasets, BrainGVEX and CommonMind, revealing 137 and 88 trait-associated exons (in 84 and 67 genes), respectively. Enriched biological functions in the associated gene sets converged on neuronal function and development, immune cell activation, and cellular transport, which are highly relevant to SCZ. SpliTWAS variants impacted RNA-binding protein binding sites, revealing potential disruption of RNA-protein interactions affecting splicing. We extended the probabilistic fine-mapping method FOCUS to the exon level, identifying 36 genes and 48 exons as putatively causal for SCZ. We highlight VPS45 and APOPT1, where splicing of specific exons was associated with disease risk, eluding detection by conventional gene expression analysis. Collectively, this study supports the substantial role of alternative splicing in shaping the genetic basis of SCZ, providing a valuable approach for future investigations in this area.
      Cover page: Splicing-specific transcriptome-wide association uncovers genetic mechanisms for schizophrenia
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2021)
      Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale data sets of the Genotype-Tissue Expression (GTEx) project to study genetically modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues versus individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMAS-associated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.
        Cover page: Allele-specific alternative splicing and its functional genetic variants in human tissues
        • 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
          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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