Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A.suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A.suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m1A9). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of aminoacylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m1A9 is a key residue for the activity of T-armless tRNAs. Thus, m1A9 is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

[1]  R Giegé,et al.  The 2.0 A crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules. , 2000, Structure.

[2]  K. Watanabe,et al.  Primary sequence of mitochondrial tRNA(Arg) of a nematode Ascaris suum: occurrence of unmodified adenosine at the first position of the anticodon. , 1997, Biochimica et biophysica acta.

[3]  O. Uhlenbeck,et al.  Synthesis of modified nucleoside 3',5'-bisphosphates and their incorporation into oligoribonucleotides with T4 RNA ligase. , 1978, Biochemistry.

[4]  Masayuki Sakurai,et al.  Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu. , 2002, Nucleic acids research.

[5]  J. A. Wahleithner,et al.  Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Watanabe,et al.  Higher-order structure of bovine mitochondrial tRNA(Phe) lacking the 'conserved' GG and T psi CG sequences as inferred by enzymatic and chemical probing. , 1994, Nucleic acids research.

[7]  R. Okimoto,et al.  The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. , 1992, Genetics.

[8]  R. Gasser,et al.  The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea). , 2002, International journal for parasitology.

[9]  Takashi Ohtsuki,et al.  A unique serine-specific elongation factor Tu found in nematode mitochondria , 2003, Nature Structural Biology.

[10]  R. Okimoto,et al.  A set of tRNAs that lack either the T psi C arm or the dihydrouridine arm: towards a minimal tRNA adaptor. , 1990, The EMBO journal.

[11]  G. Krauss,et al.  Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study. , 1977, European journal of biochemistry.

[12]  Richard Giegé,et al.  The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA. , 1998, Nucleic acids research.

[13]  H. Donis-Keller,et al.  Phy M: an RNase activity specific for U and A residues useful in RNA sequence analysis. , 1980, Nucleic acids research.

[14]  T. Ohtsuki,et al.  Stable isotope-edited NMR analysis of Ascaris suum mitochondrial tRNAMet having a TV-replacement loop. , 1998, Journal of biochemistry.

[15]  Henri Grosjean,et al.  Modification And Editing Of Rna , 1998 .

[16]  N. Hanyu,et al.  Analysis of modified nucleosides and nucleotide sequence of tRNA. , 1987, Methods in enzymology.

[17]  T. Ohtsuki,et al.  An “Elongated” Translation Elongation Factor Tu for Truncated tRNAs in Nematode Mitochondria* , 2001, The Journal of Biological Chemistry.

[18]  Tsutomu Suzuki,et al.  Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases , 2002, The EMBO journal.

[19]  J. F. Curran.  Modified Nucleosides in Translation , 1998 .

[20]  T. Unnasch,et al.  The mitochondrial genome of Onchocerca volvulus: sequence, structure and phylogenetic analysis. , 1998, Molecular and biochemical parasitology.

[21]  K. Watanabe,et al.  Primary and higher order structures of nematode (Ascaris suum) mitochondrial tRNAs lacking either the T or D stem. , 1994, The Journal of biological chemistry.

[22]  T. Ohtsuki,et al.  Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments. , 1996, Nucleic acids research.

[23]  R Giegé,et al.  A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. , 1999, Biochemistry.

[24]  L. Mosyak,et al.  The crystal structure of phenylalanyl-tRNA synthetase from thermus thermophilus complexed with cognate tRNAPhe. , 1997, Structure.

[25]  J. McCloskey,et al.  Isotopic labeling studies of the base-catalyzed conversion of 1-methyladenosine to N 6 -methyladenosine. , 1973, The Journal of organic chemistry.

[26]  Sergey Steinberg,et al.  Compilation of tRNA sequences and sequences of tRNA genes , 2004, Nucleic Acids Res..