Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

RNA. 2015 May;21(5):824-32. doi: 10.1261/rna.049197.114. Epub 2015 Mar 4.

Abstract

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3'-OH/5'-PO4 duplexes in which the 3'-OH strand is RNA. It does so via the "classic" ligase pathway, entailing reaction with ATP to form a covalent NgrRnl-AMP intermediate, transfer of AMP to the nick 5'-PO4, and attack of the RNA 3'-OH on the adenylylated nick to form a 3'-5' phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3'-OH/5'-PO4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.

Keywords: RNA repair; adenylyltransferase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain / genetics
  • Cloning, Molecular
  • Deinococcus / enzymology
  • Deinococcus / genetics
  • Molecular Sequence Data
  • Naegleria / enzymology
  • Naegleria / genetics*
  • Polynucleotide Adenylyltransferase / metabolism
  • Protein Structure, Tertiary
  • RNA Ligase (ATP) / chemistry
  • RNA Ligase (ATP) / genetics
  • RNA Ligase (ATP) / metabolism*
  • RNA Processing, Post-Transcriptional / genetics*
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism*
  • Sequence Homology

Substances

  • RNA, Protozoan
  • Polynucleotide Adenylyltransferase
  • RNA Ligase (ATP)