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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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Exon-intron border regions of unspliced forms of tRNAGlu(UUC) (A); tRNAGlu(CUC) (B); and tRNALeu(GAG) (C) of S. tokodaii strain7 [15]. These are examples of introns located at positions other than position “37/38”. In (A,B), only the D-arm region is shown; In (C), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [15]. The anticodon sequence of tRNALeu(GAG) is boxed.
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ijms-16-02187-f007: Exon-intron border regions of unspliced forms of tRNAGlu(UUC) (A); tRNAGlu(CUC) (B); and tRNALeu(GAG) (C) of S. tokodaii strain7 [15]. These are examples of introns located at positions other than position “37/38”. In (A,B), only the D-arm region is shown; In (C), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [15]. The anticodon sequence of tRNALeu(GAG) is boxed.

Mentions: Based on comparisons between sequences representing spliced and unspliced transcript pairs from interrupted tRNA genes, we could not determine the precise exon-intron borders for 13 of 24 interrupted tRNA genes in S. tokodaii strain7 because of sequence redundancy at these borders [15]. For example, the nucleotide sequences of the 3' region of the 5' exon and the 5' region of the 3' exon of tRNAGlu(UUC) (Figure 7A) are C22-A23-A24-G25-C26 and a38-a39-a40-g41-t42. There are four possibilities for the spliced sequence: C22-a39-a40-g41-t42, C22-A23-a40-g41-t42, C22-A23-A24-g41-t42, and C22-A23-A24-G25-t42. Thus it was impossible to determine the precise exon-intron border based on the sequence of the corresponding cDNA (...CAAGT...). This limitation is shared with earlier genome-wide studies of tRNAs [9]. Although the prediction of a BHB structure may indicate the putative exon-intron borders, in some cases, typical BHB structures may not be present in these molecules. Instead, variations of BHB including mismatches in helices and/or bulges of with lengths other than three residues may be predicted. For example, the A. pernix unspliced tRNALys(CUU) does not form a helix that flanks the central helix between the 5' exon and the 3' exon (Figure 4K). The crenarchaeal splicing endonuclease (EndA) could cleave these varieties of BHB-like structures [17]. We have successfully expressed a functional, recombinant S. tokodaii EndA [18] and have used it to analyze the cleavage site of some synthetic unspliced S. tokodaii tRNAs in vitro. Consequently, we could determine the precise exon-intron borders of each interrupted tRNA [15].


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

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

Exon-intron border regions of unspliced forms of tRNAGlu(UUC) (A); tRNAGlu(CUC) (B); and tRNALeu(GAG) (C) of S. tokodaii strain7 [15]. These are examples of introns located at positions other than position “37/38”. In (A,B), only the D-arm region is shown; In (C), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [15]. The anticodon sequence of tRNALeu(GAG) is boxed.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-02187-f007: Exon-intron border regions of unspliced forms of tRNAGlu(UUC) (A); tRNAGlu(CUC) (B); and tRNALeu(GAG) (C) of S. tokodaii strain7 [15]. These are examples of introns located at positions other than position “37/38”. In (A,B), only the D-arm region is shown; In (C), only the D-arm and anticodon arm regions are shown. The arrowheads indicate the exon-intron borders determined in our previous study [15]. The anticodon sequence of tRNALeu(GAG) is boxed.
Mentions: Based on comparisons between sequences representing spliced and unspliced transcript pairs from interrupted tRNA genes, we could not determine the precise exon-intron borders for 13 of 24 interrupted tRNA genes in S. tokodaii strain7 because of sequence redundancy at these borders [15]. For example, the nucleotide sequences of the 3' region of the 5' exon and the 5' region of the 3' exon of tRNAGlu(UUC) (Figure 7A) are C22-A23-A24-G25-C26 and a38-a39-a40-g41-t42. There are four possibilities for the spliced sequence: C22-a39-a40-g41-t42, C22-A23-a40-g41-t42, C22-A23-A24-g41-t42, and C22-A23-A24-G25-t42. Thus it was impossible to determine the precise exon-intron border based on the sequence of the corresponding cDNA (...CAAGT...). This limitation is shared with earlier genome-wide studies of tRNAs [9]. Although the prediction of a BHB structure may indicate the putative exon-intron borders, in some cases, typical BHB structures may not be present in these molecules. Instead, variations of BHB including mismatches in helices and/or bulges of with lengths other than three residues may be predicted. For example, the A. pernix unspliced tRNALys(CUU) does not form a helix that flanks the central helix between the 5' exon and the 3' exon (Figure 4K). The crenarchaeal splicing endonuclease (EndA) could cleave these varieties of BHB-like structures [17]. We have successfully expressed a functional, recombinant S. tokodaii EndA [18] and have used it to analyze the cleavage site of some synthetic unspliced S. tokodaii tRNAs in vitro. Consequently, we could determine the precise exon-intron borders of each interrupted tRNA [15].

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