UC Santa Cruz

RNA is central to life, acting as a bridge between genetic information and functional proteins. This dissertation explores two key aspects of RNA biology: the technical biases introduced by poly(A) selection in RNA sequencing and the mechanistic processes of Nonsense-Mediated mRNA Decay (NMD) in Caenorhabditis elegans. The first investigation uncovered that poly(A) selection skews sequencing results toward mRNAs with longer poly(A) tails, distorting our understanding of RNA populations and dynamics. This insight underscored the need for more inclusive sequencing approaches, which ultimately set the stage for applying Nanopore Sequencing to our core questions in NMD.The second focus centers on resolving the biochemical pathways by which animal cells attack NMD target mRNAs—an essential process for both cellular quality control and transcriptome regulation. Using single-molecule nanopore sequencing, we investigated the fates of NMD-targeted mRNAs, revealing that these targets undergo deadenylation and decapping at levels similar to normal mRNAs. We demonstrated that SMG-5, a protein previously implicated in deadenylation and decapping, is crucial for SMG-6-mediated endonucleolytic cleavage. Our results support a model in which NMD factors act in concert to degrade NMD targets in animals via an endonucleolytic cleavage near the stop codon, while deadenylation and decapping serve as routine aspects of typical mRNA (and NMD target mRNA) maturation and decay rather than exclusive features of NMD. Together, these studies contribute to refining RNA sequencing methodologies and deepening our understanding of NMD’s molecular mechanisms, offering insights that advance our knowledge of RNA biology.