UC Riverside

RNA processing is a fundamental biological mechanism that dictates how genetic information encoded in DNA is ultimately translated into polypeptides. This dissertation focuses on utilizing mass spectrometry to uncover key proteins involved in RNA processing under various perturbations and to unravel how these perturbations affect RNA processing through these proteins.In Chapter 2, I described a quantitative proteomics experiment using affinity pull-down with a biotin-As(III) probe to identify nuclear arsenite-binding proteins. I uncovered several candidate As(III)-binding proteins involved in mRNA splicing, DNA repair, and DNA replication. Notably, I found that As(III) binds to splicing factor 1 (SF1), perturbing mRNA splicing in human cells. This work provides insights into the mechanisms of As(III) toxicity by revealing new nuclear protein targets of As(III).In chapter 3, I utilized a scheduled LC-PRM method to quantify changes in abundances of epitranscriptomic reader, writer, and eraser (RWE) proteins in BRAFV600E melanoma cells that developed resistance to vemurafenib. My results revealed elevated expressions of MTO1 and TRMU, two RNA-modifying enzymes that act sequentially produce a single mitochondrial tRNA modification, in the resistant cell line. I found that genetic depletion of TRMU re-sensitizes resistant cells to vemurafenib and reduces mitochondrial respiration, highlighting the vital role of a mitochondrial tRNA-modifying enzyme in conferring vemurafenib resistance in melanoma.In Chapter 4, I applied the LC-PRM method to assess how genetic depletion of TRUB1, a pseudouridine synthase that targets Ψ55 on tRNA, affects the expression levels of other epitranscriptomic RWE proteins. We detected a 40% reduction of TARBP1 level in TRUB1 knockout (KO) cells. TARBP1 is a methyltransferase that deposits 2'-O-methylation on guanosine at the 18th position in tRNA (Gm18). Exhaustive digestion of tRNA followed by LC-MS/MS showed about a 50% reduction of Gm levels in TRUB1 KO cells. This result suggests that TRUB1 KO might regulate Gm levels by down-regulating TARBP1.In summary, this dissertation involves the application of mass spectrometry to identify key proteins involved in RNA processing and the investigation about how environmental perturbations alter the RNA landscape through these proteins. These findings highlight the significant regulatory role of these RNA-processing proteins and illuminate their intricate involvement in modulating cellular processes.