title: Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia.
abstract: A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA(Tyr I). Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5'-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA.
authors.forename: Balasubrahmanym
authors.initials: B
authors.lastname: Addepalli
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA Department of Chemistry, The College of Wooster, Wooster, Ohio 44691-2393, USA.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Lesner
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D029777:Momordica charantia / Q000201:enzymology*; D012323:RNA Processing, Post-Transcriptional; D018749:RNA, Plant / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014529:Uridine / Q000378:metabolism*
chemical_list: D018749:RNA, Plant; D012260:Ribonucleases; C413748:ribonuclease MC1; D014529:Uridine
keywords: LC MS/MS; RNA modification mapping; RNA sequencing; RNA structure; enzymatic cleavage; modified nucleosides; pseudouridine; uridine