UC San Diego

All RNAs in the cell are monitored by robust molecular machinery that ensures its fidelity and quality. Maintenance of the integrity of the transcriptome is vital to the proper functioning of the cell. Dysfunctions in the machinery responsible for this quality control can lead to physiological problems for the cell and wide-ranging diseases in the context of a whole organism. A deeper understanding of these systems is critically important to illuminate the workings of the cell, multi-cellular organisms, and to provide new therapeutic handles to treat human disease. The cell contains multiple overlapping quality control pathways to monitor RNAs that are tailored to the type and purpose of a particular RNA. RNAs that do not code for proteins, non-coding RNAs (ncRNAs), are increasingly being understood for the dynamic trimming and tailing that occurs in their 3’ end to control their maturation and quality. Advances in next generation sequencing have allowed researchers to examine the changing 3’ ends of ncRNAs at nucleotide resolution, but robust, open source, and publicly available tools to analyze this data have been missing from the field. In chapter 2 of this dissertation, I outline a bioinformatic tool dubbed “Tailer” that allows for visualization and quantification of this kind of data. This tool is thoroughly validated on public datasets and faithfully recapitulates the findings of the original studies from which they were drawn. Another key hub for RNA quality control, specifically for mRNAs, is translation. When translating a dysfunctional mRNA or an mRNA in a difficult context, a ribosome can run into regions that cause a local ribosome stall. This allows a trailing ribosome to collide, signaling to the cell that the nascent protein and mRNA template need to be degraded. In chapter 3, I present evidence that Angel1, a 2’,3’ cyclic phosphatase, is a novel rate-limiting factor for this ribosome-associated quality control (RQC) pathway. Angel1 associates with proteins important for this process and nucleotide sequences that have been implicated in ribosome stalling. Angel1 depletion also stabilizes reporter substrates that are targets of this pathway. I also show evidence that N4BP2 is the human ortholog of the RQC endonuclease identified in Saccharomyces cerevisiae and Caenorhabditis elegans. Lastly, in chapter 4, I examine Angel2, a protein homologous to Angel1, that was recently identified as a 2’,3’ cyclic phosphatase. Using discovery-based techniques, I look for clues that could suggest Angel2’s function. With IP-MS/MS I find that Angel2 associates with components of the tRNA splicing ligase. Using eCLIP, I find evidence that Angel2 directly associates with tRNAs but is not necessarily enriched for the subset of tRNAs that undergo splicing. Finally, with RNA-seq, I find that Angel2 depletion upregulates mRNAs with a low fraction of rare codons and codons decoded by intron-containing tRNAs. These studies provide new insights into RNA quality control and provide a leaping off point for future studies into the modification and processing of RNA 3' ends.