Mechanism of U-insertion RNA editing in trypanosome mitochondria: characterization of RET2 functional domains by mutational analysis.
3'-Terminal uridylyl transferases (TUTases) selectively bind uridine 5'-triphosphate (UTP) and catalyze the addition of uridine 5'-monophosphate to the 3'-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.
Ringpis
Gene-Errol
GE
Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, B240 Medical Sciences I, Irvine, CA 92697, USA.
Stagno
Jason
J
Aphasizhev
Ruslan
R
D001665:Binding Sites; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D014345:Trypanosoma / Q000201:enzymology* / Q000235:genetics; D014544:Uridine Triphosphate / Q000378:metabolism
D015800:Protozoan Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; C029305:mitochondrial messenger RNA; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D014544:Uridine Triphosphate