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Experimental confirmation of a whole set of tRNA molecules in two archaeal species.

Watanabe Y, Kawarabayasi Y - Int J Mol Sci (2015)

Bottom Line: To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers.Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule.Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

View Article: PubMed Central - PubMed

Affiliation: University of Tokyo, Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. ywatanab@m.u-tokyo.ac.jp.

ABSTRACT
Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

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Summary of the tRNA introns identified in S. tokodaii strain7 [15]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.
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ijms-16-02187-f005: Summary of the tRNA introns identified in S. tokodaii strain7 [15]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.

Mentions: A thermoacidophilic archaeon, S. tokodaii strain7, was isolated from an acidic spa in Beppu Hot Springs in Kyushu, Japan. This microorganism is aerobic and grows optimally at 80 °C and pH 2.5–3 [11]. S. tokodaii strain7 belongs to the order Sulfoflobales, Crenarcaheota, and not the order Desulfolococcales, to which A. pernix K1 belongs [6]. The entire genomic sequence of this Crenarchaeon was published in 2001 [12]. The length of the genome is 2,694,756 bp with a G + C content of approximately 33%. This sequence is predicted to encode approximately 2800 ORFs and one rRNA gene cluster encoding 16S and 23S rRNAs. The computer software tRNAscan [8,13] and similarity search on this genome identified a complement of 46 predicted tRNA genes including 24 predicted interrupted tRNA genes, [12]. Therefore, S. tokodaii strain7 is apparently richer in tRNA introns than is A. prenix K1. A cluster of six predicted tRNA genes was identified. Of the 46 predicted tRNA genes, 44 lack a canonical 3' CCA sequence, which is usually present in bacterial and archaeal tRNA genes [14]. Among the 24 interrupted tRNA genes predicted, three (two tRNALyss and tRNALeu(GAG)) are predicted to possess an intron in the D-arm and the anticodon stem, respectively, while other interrupted tRNA genes have the putative intron at position “37/38” (Table 2, Figure 5) [12].


Experimental confirmation of a whole set of tRNA molecules in two archaeal species.

Watanabe Y, Kawarabayasi Y - Int J Mol Sci (2015)

Summary of the tRNA introns identified in S. tokodaii strain7 [15]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307357&req=5

ijms-16-02187-f005: Summary of the tRNA introns identified in S. tokodaii strain7 [15]. Each label includes the tRNA species, the respective intron position relative to the 5' end of respective tRNA, and the lengths of the introns. Characters within parentheses indicate the nucleotide one base 5' from the intron border.
Mentions: A thermoacidophilic archaeon, S. tokodaii strain7, was isolated from an acidic spa in Beppu Hot Springs in Kyushu, Japan. This microorganism is aerobic and grows optimally at 80 °C and pH 2.5–3 [11]. S. tokodaii strain7 belongs to the order Sulfoflobales, Crenarcaheota, and not the order Desulfolococcales, to which A. pernix K1 belongs [6]. The entire genomic sequence of this Crenarchaeon was published in 2001 [12]. The length of the genome is 2,694,756 bp with a G + C content of approximately 33%. This sequence is predicted to encode approximately 2800 ORFs and one rRNA gene cluster encoding 16S and 23S rRNAs. The computer software tRNAscan [8,13] and similarity search on this genome identified a complement of 46 predicted tRNA genes including 24 predicted interrupted tRNA genes, [12]. Therefore, S. tokodaii strain7 is apparently richer in tRNA introns than is A. prenix K1. A cluster of six predicted tRNA genes was identified. Of the 46 predicted tRNA genes, 44 lack a canonical 3' CCA sequence, which is usually present in bacterial and archaeal tRNA genes [14]. Among the 24 interrupted tRNA genes predicted, three (two tRNALyss and tRNALeu(GAG)) are predicted to possess an intron in the D-arm and the anticodon stem, respectively, while other interrupted tRNA genes have the putative intron at position “37/38” (Table 2, Figure 5) [12].

Bottom Line: To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers.Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule.Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

View Article: PubMed Central - PubMed

Affiliation: University of Tokyo, Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. ywatanab@m.u-tokyo.ac.jp.

ABSTRACT
Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

Show MeSH