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Ribonucleic acids (RNAs) are challenging structural biology targets, as numerous barriers exist to determining their high-resolution structures and specific biological functions. Previous results have highlighted the utility of collision-induced unfolding (CIU) to relatively rapidly assess noncoding (nc)RNA higher-order structure (HOS) information. Yet, there remain many gaps in our understanding of how these data can be related to the structures adopted by RNAs in solution as current correlations are largely qualitative. In this study, we describe significant advancements in RNA CIU. Previous RNA CIU reports reveal minimal-to-no RNA unfolding events (or features) upon being subjected to standard CIU conditions. Here, we increase the RNA CIU information through supercharging and quantitatively evaluate the improved RNA CIU data obtained to solution-phase unfolding data collected across a range of Mg2+ concentrations. Finally, we apply our supercharged CIU experiment to mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS)-associated mt-tRNA leucine (Leu, UUR) (mt-tRNALeu(UUR)) species. Our data demonstrate strong quantitative correlations between gas-phase and solution-phase RNA unfolding events as a function of Mg2+ and MELAS-associated mutations. Taken together, these results indicate strong, solution-relevant relationships for CIU data collected for these RNAs. We conclude our work by discussing future work targeting RNA CIU annotation, broader biophysical characterization of disease-associated RNAs using CIU, and CIU-enabled transcriptomic analysis. 

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing their applications in biotechnology. The inherent dynamism of RNAs creates numerous obstacles in capturing their biologically relevant higher-order structures (HOSs), and as a result, many RNA functions remain unknown. In this study, we describe the development of native ion mobility–mass spectrometry and collision-induced unfolding (CIU) for the structural characterization of a variety of RNAs. We evaluate the ability of these techniques to preserve native structural features in the gas phase across a wide range of functional RNAs. Finally, we apply these tools to study the elusive mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNAs via the correlated complexity of CIU fingerprints and RNA HOS, the observation of predicted stability shifts in the control RNA samples, and the retention of predicted magnesium binding events in gas-phase RNA ions. Significant differences in collision cross section and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial tRNA maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization. 

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N 2-methylguanosine, N 2, N 2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N 2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.