Analysis of RNA and Its Modifications.

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins, and modulation of many biochemical processes. They are also often the key components of viruses. Synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics. In many cases, RNAs will be chemically modified, either naturally via enzymatic systems within a cell or intentionally during their synthesis. Analytical methods to detect, sequence, identify, and quantify RNA and its modifications have demands that far exceed requirements found in the DNA realm. Two complementary platforms have demonstrated their value and utility for the characterization of RNA and its modifications: mass spectrometry and next-generation sequencing. This review highlights recent advances in both platforms, examines their relative strengths and weaknesses, and explores some alternative approaches that lie at the horizon.

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides.

Oxidative stress triggered by the Fenton reaction (chemical) or UVR exposure (photo) can damage cellular biomolecules including RNA through oxidation of nucleotides. Besides such xenobiotic chemical modifications, RNA also contains several post-transcriptional nucleoside modifications that are installed by enzymes to modulate structure, RNA-protein interactions, and biochemical functions. We examined the extent of oxidative damage to naturally modified RNA which is required for cellular protein synthesis under two different contexts. The extent of oxidative damage is higher when RNA is not associated with proteins, but the degree of damage is lower when the RNA is presented in the form of a ribonucleoprotein complex, such as an intact ribosome. Our studies also indicate that absence of methylations in ribosomal RNA at specific positions could make it more susceptible to photooxidative stress. However, the extent of guanosine oxidation varied with the position at which the modification is deficient, indicating position-dependent structural effects. Further, an E. coli strain deficient in 5-methylaminomethyl-2-thiouridine (mnm5s2U) (found in lysine and glutamate tRNA anticodon) is more vulnerable to oxidative RNA damage compared to its wildtype strain suggesting an auxiliary function for the mnm5s2U modification. These studies indicate that oxidative damage to RNA is altered by the presence of enzymatic modified nucleosides or protein association inside the cell.