Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant

Abstract

We report the identification and use of a mutant of the purine selective ribonuclease RNase U2 that randomly cleaves RNA in a manner that is directly compatible with RNA modification mapping by mass spectrometry. A number of RNase U2 mutants were generated using site-saturation mutagenesis. The enzyme activity and specificity were tested using oligonucleotide substrates, which revealed an RNase U2 E49A mutant with limited specificity and a tendency to undercut RNA. Using this mutant, RNA digestion conditions were optimized to yield long, overlapping digestion products, which improve sequence coverage in RNA modification mapping experiments. The analytical utility of this mutant was demonstrated by liquid chromatography tandem mass spectrometry (LC-MS/MS) mapping of several modified RNAs where 100% sequence coverage could be obtained using only a single enzymatic digestion. This new mutant facilitates more accurate and efficient RNA modification mapping than traditional highly base-specific RNases that are currently used.

tRNA Metabolism and Neurodevelopmental Disorders

“…The most accepted and accurate approach to mapping tRNA modifications is liquid chromatography tandem mass spectrometry (87, 140). RNase enzymes have been engineered to enable complete coverage with a single enzymatic digestion (169). A recent alternative approach to measuring tRNA modification levels at nucleotide resolution is comparative methylation analysis through AlkB-facilitated RNA methylation sequencing (ARM-seq) (32), although this technique is currently limited to methylation at tRNA positions accessible to the AlkB enzyme.…”

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

“…Oligonucleotides of length 5–15 nucleotides are desirable as this length will be unique yet small enough to decrease the complexity of data analysis. In recent years, there has been a push to identify and implement new RNases that can be used in combination to increase sequence coverage through the generation of overlapping digestion products [ 103 , 104 , 105 ]. However, as these RNases have been produced in-house in the labs that develop them, they suffer from a lack of reproducibility.…”

The epitranscriptome toolbox

“…In principle, LC-MS/MS can also be used to map RNA modifications in RNA fragments to provide a site-specific context (Solivio et al, 2018). However, this method requires fine adjustment of the fragmentation step to ensure transcript breakdown into short overlapping fragments and hundreds of nanograms of each transcript, making it unsuitable for analysis of mRNAs, due to the relatively low abundance of individual transcripts and the large diversity in the transcriptome (Solivio et al, 2018).…”