Consequences Of Uridine mRNA Modifications On Translation

Abstract

Messenger RNAs play a crucial role as a road map for the ribosome to decode during protein synthesis. In the past decade, it was discovered that a small subset of modifications can be post-transcriptionally incorporated into messenger RNA (mRNA) sequences responsible for directing protein synthesis. In addition, non-coding RNAs have long been known to contain chemical modifications that are recognized as key modulators of their structure and function in cells. The enzymatic incorporation of mRNA modifications has the potential to influence every step of the mRNA lifecycle including, mRNA stability, protein recruitment, and translation. This collection of work focuses on the consequences that incorporation of these modification have on mRNA translation and tRNA function, specifically uridine modifications. First, I investigated how one of the most common mRNA modifications, pseudouridine, impacts mRNA translation. We first discovered that pseudouridine has the ability to alter amino addition rates, decrease translation fidelity, alter GTP hydrolysis rate and has a distal effect on the CCA 3’ end of tRNAphe when present at the first position of a UUU codon. We then explored the newly discovered uridine modification, 5-methyluridine (m5U) also known as ribothymidine. Using LC-MS/MS techniques we not only discovered multiple methylated mRNA modifications, but were also able to identify the enzymes that incorporated them. In this work, it was revealed that 5-methyluridine is incorporated into yeast mRNAs by the tRNA (uracil-5-)-methyltransferase 2 enzyme (Trm2). I also revealed that the m5U modification has different effects on mRNA translation and tRNA role in the translation process respectively. Interestingly, both pseudouridine and m5U show that context is important when displaying a specific phenotype, each acting in a position dependent manner regarding the 1st, 2nd, or 3rd position in a codon. In this dissertation, I was specifically interested in investigating how mRNA modifications impact translation. I was able to highlight that each modification plays specific and distinct roles in how it affects mRNA translation, and that it is important for the field to characterize them individually. The scope of this research surpasses merely understanding the mechanist impacts modifications has on translation, but open avenues of exploration into drug resistance, therapeutics, protein evolution, and the overall cellular stress response. In the past decade, a new field of therapeutics directed around mRNA emerged. This process was particularly streamlined to commercial availability by the COIVD-19 pandemic. In fact, both the Moderna and Pfizer mRNA vaccines contain modified mRNA by adding N1-methylpseudouridine; which helps with both mRNA stability and immune response. Therefore, understanding how these modifications function could help incorporate more of them into mRNA therapeutics, and help create better treatments. In addition, it is newly discovered that tRNA modifications may have a function in antibiotic resistance, making the modifying enzymes associated with them great drug targets. This is further supported by my work showing that knocking out Trm2 changes the response to translation inhibiting antibiotics. Furthermore, my work implies that these uridine modifications function may be linked to cellular stress responses. For pseudouridine specifically, it is known it increases during stress, and it is hypothesized that this could be a function of pseudouridine itself. In fact, this coupled with my research, could show that pseudouridine is incorporated into mRNA to allow for small miscoding events create alternative proteins during stress and/or promote protein evolution.