Eukaryotic gene expression from DNA to protein requires multiple RNA maturation steps including 5′ end capping, splicing, and 3′ end cleavage and polyadenylation. In most eukaryotic organisms, genes produce multiple mRNA isoforms by way of alternative splicing, alternative 3′ end maturation, and use of alternative transcriptional start sites. Alternative splicing in particular is a powerful mechanism for expanding gene function through both the protein coding capacity and regulatory potential of individual mRNA molecules. RNA binding proteins (RBPs) recruited to the mRNA during or as a consequence of splicing couple the nuclear history of the message to its cytoplasmic fate. In this thesis I explore how alternative mRNA processing and differential mRNP composition influence gene expression.
Simple measures of steady-state RNA levels by RNA-Seq do not always correlate with ultimate protein output. In some tissues, and for different classes of genes, the relationship appears to be under developmental control, suggesting that mRNA is being repressed at the translational level. Using mass spectrometry to detect relative expression of two protein isoforms from the same gene is extremely difficult since the alternative protein sequence is a small part of the entire protein. To address this problem we assess global mRNA isoform-specific ribosome association that preserves the entirety of the message (Frac-Seq) as a direct and independent signal of the extent to which each isoform engages with translational machinery. We explore alternative isoform ribosome association in two contexts: 1) in a comparative transcriptomics study, where we are able to leverage millions of years of random “experiments” in the form of evolutionary changes in primate genome sequence; and 2) in inchoate human neural differentiation, where alternative splicing is abundant and RNA-protein correlation is particularly low. All together, these investigations will help us understand how alternative mRNA processing is important for accurate and efficient gene expression.
In addition, in the middle of March 2020, the COVID-19 pandemic suddenly shut down our ability to do research. Uncertainty has been a major theme since then. A leading contributor to personal and public uncertainties was the lack of reliable testing for SARS-CoV-2. A group of UCSC students, staff, and faculty realized we had the technical knowledge to help keep our university community safe. Under the FDA's Emergency Use Authorization (EUA) orders and our Student Health Center's clinical laboratory credentials, we implemented a molecular biology-based SARS-Cov-2 diagnostic test and a campus-wide COVID-19 surveillance program. This initiative has been instrumental in breaking SARS-Cov-2 transmission chains on campus and in the broader community and had an immediate impact on human health and safety. A roadmap for establishing a “pop-up” clinical molecular biology-based diagnostics laboratory is included as a chapter.
