Search

Proper regulation of gene expression is critical for organismal life and is dictated by numerous complex transcriptional and post-transcriptional regulatory pathways. Profiling the transcriptome by sequencing to assess gene expression is a powerful approach that facilitates understanding these modes of regulation. This dissertation explores gene expression and post- transcriptional regulation in numerous contexts.Chapter 2 summarizes my in-depth analysis of RNA expression and regulation in aging C. elegans, where we leveraged the Oxford Nanopore Technologies platform to perform full- length direct RNA sequencing in tandem with short-read sequencing. I investigated changes to gene expression that occur during aging and identified 814 novel transcript isoforms. I report an age-dependent increase in poly(A) tail lengths genome-wide and a decrease in splice site fidelity. I also leveraged these methods to examine pseudouridine and inosine modified RNA nucleotides and found that there are more pseudouridine sites in old animals compared to young. Together, these results forge a deeper understanding of age-dependent transcriptomic changes that may contribute to degenerative aging phenotypes.In Chapter 3, I summarize work where I improved tools to conditionally deplete microRNA Argonuate proteins, ALG-1 and ALG-2, which are important post-transcriptional regulators that downregulate target RNA expression. Unexpectedly, depletion of these proteins does not elicit upregulation of target RNAs, which I report and explore. In Chapter 4 I report limitations of the original auxin inducible degron (AID) system, which I improved upon in Chapter 3. In its original iteration, we observed basal degradation of AID-tagged proteins, which limited its applications. Chapter 5 is an examination of poly(A) tail lengths in Influenza A virus (IAV), which uses a non-canonical mode of polyadenylation. We observed that poly(A) tail lengths of IAV are longer than predicted and are subject to shortening over the course of viral infection.