Jump to main content
Jump to site search


Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

Author affiliations

Abstract

Locating ribonucleoside modifications within an RNA sequence requires digestion of the RNA into oligoribonucleotides of amenable size for subsequent analysis by LC-MS (liquid chromatography-mass spectrometry). This approach, widely referred to as RNA modification mapping, is facilitated through ribonucleases (RNases) such as T1 (guanosine-specific), U2 (purine-selective) and A (pyrimidine-specific) among others. Sequence coverage by these enzymes depends on positioning of the recognized nucleobase (such as guanine or purine or pyrimidine) in the sequence and its ribonucleotide composition. Using E. coli transfer RNA (tRNA) and ribosomal RNA (rRNA) as model samples, we demonstrate the ability of complementary nucleobase-specific ribonucleases cusativin (C-specific) and MC1 (U-specific) to generate digestion products that facilitate confident mapping of modifications in regions such as G-rich and pyrimidine-rich segments of RNA, and to distinguish C to U sequence differences. These enzymes also increase the number of oligonucleotide digestion products that are unique to a specific RNA sequence. Further, with these additional RNases, multiple modifications can be localized with high confidence in a single set of experiments with minimal dependence on the individual tRNA abundance in a mixture. The sequence overlaps observed with these complementary digestion products and that of RNase T1 improved sequence coverage to 75% or above. A similar level of sequence coverage was also observed for the 2904 nt long 23S rRNA indicating their utility has no dependence on RNA size. Wide-scale adoption of these additional modification mapping tools could help expedite the characterization of modified RNA sequences to understand their structural and functional role in various living systems.

Graphical abstract: Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

Back to tab navigation

Supplementary files

Article information


Submitted
22 Oct 2019
Accepted
02 Dec 2019
First published
02 Dec 2019

Analyst, 2020, Advance Article
Article type
Paper

Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

P. Thakur, M. Estevez, P. A. Lobue, P. A. Limbach and B. Addepalli, Analyst, 2020, Advance Article , DOI: 10.1039/C9AN02111F

Social activity

Search articles by author

Spotlight

Advertisements