RtcB Is the RNA Ligase Component of an Escherichia coli RNA Repair Operon

Naoko Tanaka,Stewart Shuman

doi:10.1074/jbc.c111.219022

Abstract

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.

Highlights

Programmed breakage of the tRNA anticodon loop by sitespecific endoribonucleases is an ancient mechanism by which microbes either respond to virus infection or distinguish self from non-self [1,2,3,4,5]. tRNA ribotoxins employ a transesterification mechanism that generates 5Ј-OH and 2Ј,3Јcyclic phosphate termini
In considering alternative RNA repair strategies, our attention has focused on E. coli RNA 3Ј-phosphate cyclase (RtcA) as a candidate RNA repair enzyme
Recent studies revealed that the catalytic repertoire of RNA cyclase overlaps that of RNA/DNA ligases; to wit, E. coli RtcA catalyzes adenylylation of 5Ј-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products [39]

Summary

Introduction

Programmed breakage of the tRNA anticodon loop by sitespecific endoribonucleases (ribotoxins) is an ancient mechanism by which microbes either respond to virus infection or distinguish self from non-self [1,2,3,4,5]. tRNA ribotoxins employ a transesterification mechanism that generates 5Ј-OH and 2Ј,3Јcyclic phosphate termini. If RtcA is involved directly in an RNA-sealing pathway, it either requires a protein partner (ala Pnkp-Hen1) or plays a supportive role in RNA repair by providing a 2Ј,3Ј-cyclic phosphate substrate for a separate bacterial RNA ligase enzyme.

Results

Conclusion