Improved RNA‐modification mapping through employment of novel ribonucleases and LC‐MS

Abstract

More than 150 different chemical modifications have been documented in various types of ribonucleic acids (RNA) [1]. They play key roles in translation, cellular fate, and stabilization of RNA structure [2]. While majority of these modifications are catalyzed by cellular enzymes, others are generated by xenobiotic stress exposure. RNA modifications have been linked to human diseases [3] such as neurological disorders and cancer.Understanding the role of these modifications in human disorders requires precise identification and location of the site in the overall RNA sequence. Nucleobase specific ribonuclease (RNase) enzymes are useful in mapping the location of chemical modification. Here the RNA is digested with a nucleobase‐specific enzyme and the resulting digestion products are subjected to liquid chromatography coupled with mass spectrometry (LC‐MS) ‐based sequencing, a method widely referred to as RNA modification mapping [4].Commercially available RNase T1 (G‐specific) and RNase A (pyrimidine‐specific) have been widely used in RNA modification mapping procedures. To augment the limitations of these enzymes, additional nucleobase‐specific enzymes, RNase MC1 (U‐specific) [5] and cusativin (C‐specific) [6] have been developed that could complement the existing enzymes. Thus, the digestion products generated by multiple enzymes can complement each other thereby improving the sequence coverage and generation of overlapping digestion products of substrate RNA. In the current investigation, we will present reconstruction of modified tRNA sequences from the digestion products generated by digestion of Escherichia coli tRNA by multiple nucleobase‐specific enzymes. We will also demonstrate the utility of this technology to map the stress‐induced damage in the ribosomal RNA. The advantage of such a multi‐pronged approach as opposed to the single enzyme usage in reconstruction of modified RNA sequences will be discussed.Support or Funding InformationFinancial support for this work was provided by the National Institutes of Health (GM058843) and the University of Cincinnati.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.