Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi.
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.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Mihaela-Carmen
MC
Unciuleac
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Stewart
S
Shuman
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009253:Naegleria / Q000201:enzymology / Q000235:genetics*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011130:RNA Ligase (ATP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D017385:Sequence Homology
D016053:RNA, Protozoan; D011062:Polynucleotide Adenylyltransferase; D011130:RNA Ligase (ATP)
RNA repair; adenylyltransferase