TY - JOUR
T1 - Nematode-specific tRNAs that decode an alternative genetic code for leucine
AU - Hamashima, Kiyofumi
AU - Fujishima, Kosuke
AU - Masuda, Takeshi
AU - Sugahara, Junichi
AU - Tomita, Masaru
AU - Kanai, Akio
N1 - Funding Information:
The N2 nematode strain and Escherichia coli strain OP50 used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Total RNA was isolated from mixed-stage C. elegans, including eggs, larvae 1–4 and adults, using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. For acid–urea polyacrylamide gel electrophoresis (PAGE)/northern blot analysis, the isolated RNA was incubated with 0.2 M Tris–HCl (pH 9.5) at 37°C for 2 h to deacylate the charged tRNAs.
Funding Information:
Funding for open access charge: Grant-in-Aid for Scientific Research (B) #22370066 from the Ministry of Education, Culture, Sports, Science and Technology of Japan; research fund of the Institute for Fermentation, Osaka, Japan; research funds of the Yamagata Prefectural Government and Tsuruoka City, Japan.
PY - 2012/4
Y1 - 2012/4
N2 - Class II transfer RNAs (tRNAs), including tRNALeu and tRNA Ser, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA Gly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.
AB - Class II transfer RNAs (tRNAs), including tRNALeu and tRNA Ser, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA Gly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.
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U2 - 10.1093/nar/gkr1226
DO - 10.1093/nar/gkr1226
M3 - Article
C2 - 22187151
AN - SCOPUS:84860364684
SN - 0305-1048
VL - 40
SP - 3653
EP - 3662
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 8
ER -