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Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Muller S, Urban A, Hecker A, Leclerc F, Branlant C, Motorin Y - Nucleic Acids Res. (2009)

Bottom Line: Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy Université, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.

ABSTRACT
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

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Test of the activity of Sso and Pab cellular extracts on the Sso pre-tRNATyr(GUA) and Pab tRNATyr(GUA). The [α-32P]ATP-labelled Sso pre-tRNATyr (GUA) and Pab tRNATyr(GUA) together with their U35C variants were incubated for 90 min at 65°C in the presence of a Sso or Pab cellular extracts. Formation of residue Ψ35 was tested as described in the legend to Figure 2B, by 2D-TLC analysis after digestion of the RNA by the RNase T2. The molar amounts of Ψ residue formed per mole of RNA are indicated on the autoradiograms of the five 2D-TLCs. Additional spots visible on the 2D-TLC autoradiograms correspond to formation of methylated nucleotides naturally present in many archaeal tRNAs (m1G37, Cm56, m1I57 and m1A58). Reproducible results were obtained in three independent experiments.
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Figure 4: Test of the activity of Sso and Pab cellular extracts on the Sso pre-tRNATyr(GUA) and Pab tRNATyr(GUA). The [α-32P]ATP-labelled Sso pre-tRNATyr (GUA) and Pab tRNATyr(GUA) together with their U35C variants were incubated for 90 min at 65°C in the presence of a Sso or Pab cellular extracts. Formation of residue Ψ35 was tested as described in the legend to Figure 2B, by 2D-TLC analysis after digestion of the RNA by the RNase T2. The molar amounts of Ψ residue formed per mole of RNA are indicated on the autoradiograms of the five 2D-TLCs. Additional spots visible on the 2D-TLC autoradiograms correspond to formation of methylated nucleotides naturally present in many archaeal tRNAs (m1G37, Cm56, m1I57 and m1A58). Reproducible results were obtained in three independent experiments.

Mentions: As no Ψ35 had yet been detected in archaeal pre-tRNATyr(GUA) or tRNATyr(GUA), the first question was to know whether such modification can occur in archaea. We failed to analyse directly the cellular Sso tRNATyr(GUA) by CMCT treatment in total RNA, because of the difficulty to find an efficient and specific primer for extension analysis. Therefore, we tested whether a pre-tRNATyr(GUA):Ψ35-synthase activity exists in S. solfataricus extracts. To this end, the in vitro transcribed Sso pre-tRNATyr(GUA) and its U35C variant, both labelled by [α-32P] ATP incorporation, were incubated for 90 min at 65°C in a S. solfataricus cellular extract. Then, by using the same approaches as above, namely RNase T2 digestion followed by 2D thin-layer chromatography, we detected the formation of 0.60 mol of Ψ residue per mole of RNA in the WT pre-tRNA, whereas no modification was detected in the U35C pre-tRNA variant (Figure 4, top panels). Formation of a Ψ residue at position 35 in the WT Sso pre-tRNATyr substrate in the Sso extract was also verified by CMCT-RT analysis (data not shown). Importantly, when using this extract, no Ψ formation was detected in the heterologous P. abyssi (Pab) tRNATyr(GUA), which is naturally synthesized without intron (Figure 4). In contrast, when the same kind of experiment was performed using a P. abyssi cellular extract and the in vitro transcribed WT or U35C variant Pab tRNATyr(GUA), 0.77 mol of Ψ residues was formed per mole of WT Pab tRNATyr(GUA) and this amount was reduced to 0.42 mol of Ψ/mol of RNA after U35C substitution (Figure 4, bottom panels). Presence of spots corresponding to several modified residues others than Ψ residues, in particular m1G37, Cm56, m1I57 and m1A58 were detected in these experiments performed with total cellular extracts. This was an indication for the high quality of these extracts in terms of RNA modification activities. Hence, taken together, the data demonstrated that a specific pre-tRNATyr(GUA):Ψ35-synthase activity exists in S. solfataricus, while in P. abyssi cells, at least one or more RNA:Ψ-synthases capable to modify tRNATyr at position 35 and at one or more positions are present (one of the possible additional modified position in this tRNA was position 13). Although we did not prove directly the presence of Ψ35 residues in tRNAsTyr extracted from P. abyssi and S. solfataricus, we demonstrated the presence of catalysts able to achieve this modification in cellular extracts from these two species. This was a strong indication for the presence of a tRNA:Ψ35-synthase activity in these two species.Figure 4.


Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Muller S, Urban A, Hecker A, Leclerc F, Branlant C, Motorin Y - Nucleic Acids Res. (2009)

Test of the activity of Sso and Pab cellular extracts on the Sso pre-tRNATyr(GUA) and Pab tRNATyr(GUA). The [α-32P]ATP-labelled Sso pre-tRNATyr (GUA) and Pab tRNATyr(GUA) together with their U35C variants were incubated for 90 min at 65°C in the presence of a Sso or Pab cellular extracts. Formation of residue Ψ35 was tested as described in the legend to Figure 2B, by 2D-TLC analysis after digestion of the RNA by the RNase T2. The molar amounts of Ψ residue formed per mole of RNA are indicated on the autoradiograms of the five 2D-TLCs. Additional spots visible on the 2D-TLC autoradiograms correspond to formation of methylated nucleotides naturally present in many archaeal tRNAs (m1G37, Cm56, m1I57 and m1A58). Reproducible results were obtained in three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651775&req=5

Figure 4: Test of the activity of Sso and Pab cellular extracts on the Sso pre-tRNATyr(GUA) and Pab tRNATyr(GUA). The [α-32P]ATP-labelled Sso pre-tRNATyr (GUA) and Pab tRNATyr(GUA) together with their U35C variants were incubated for 90 min at 65°C in the presence of a Sso or Pab cellular extracts. Formation of residue Ψ35 was tested as described in the legend to Figure 2B, by 2D-TLC analysis after digestion of the RNA by the RNase T2. The molar amounts of Ψ residue formed per mole of RNA are indicated on the autoradiograms of the five 2D-TLCs. Additional spots visible on the 2D-TLC autoradiograms correspond to formation of methylated nucleotides naturally present in many archaeal tRNAs (m1G37, Cm56, m1I57 and m1A58). Reproducible results were obtained in three independent experiments.
Mentions: As no Ψ35 had yet been detected in archaeal pre-tRNATyr(GUA) or tRNATyr(GUA), the first question was to know whether such modification can occur in archaea. We failed to analyse directly the cellular Sso tRNATyr(GUA) by CMCT treatment in total RNA, because of the difficulty to find an efficient and specific primer for extension analysis. Therefore, we tested whether a pre-tRNATyr(GUA):Ψ35-synthase activity exists in S. solfataricus extracts. To this end, the in vitro transcribed Sso pre-tRNATyr(GUA) and its U35C variant, both labelled by [α-32P] ATP incorporation, were incubated for 90 min at 65°C in a S. solfataricus cellular extract. Then, by using the same approaches as above, namely RNase T2 digestion followed by 2D thin-layer chromatography, we detected the formation of 0.60 mol of Ψ residue per mole of RNA in the WT pre-tRNA, whereas no modification was detected in the U35C pre-tRNA variant (Figure 4, top panels). Formation of a Ψ residue at position 35 in the WT Sso pre-tRNATyr substrate in the Sso extract was also verified by CMCT-RT analysis (data not shown). Importantly, when using this extract, no Ψ formation was detected in the heterologous P. abyssi (Pab) tRNATyr(GUA), which is naturally synthesized without intron (Figure 4). In contrast, when the same kind of experiment was performed using a P. abyssi cellular extract and the in vitro transcribed WT or U35C variant Pab tRNATyr(GUA), 0.77 mol of Ψ residues was formed per mole of WT Pab tRNATyr(GUA) and this amount was reduced to 0.42 mol of Ψ/mol of RNA after U35C substitution (Figure 4, bottom panels). Presence of spots corresponding to several modified residues others than Ψ residues, in particular m1G37, Cm56, m1I57 and m1A58 were detected in these experiments performed with total cellular extracts. This was an indication for the high quality of these extracts in terms of RNA modification activities. Hence, taken together, the data demonstrated that a specific pre-tRNATyr(GUA):Ψ35-synthase activity exists in S. solfataricus, while in P. abyssi cells, at least one or more RNA:Ψ-synthases capable to modify tRNATyr at position 35 and at one or more positions are present (one of the possible additional modified position in this tRNA was position 13). Although we did not prove directly the presence of Ψ35 residues in tRNAsTyr extracted from P. abyssi and S. solfataricus, we demonstrated the presence of catalysts able to achieve this modification in cellular extracts from these two species. This was a strong indication for the presence of a tRNA:Ψ35-synthase activity in these two species.Figure 4.

Bottom Line: Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy Université, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.

ABSTRACT
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

Show MeSH
Related in: MedlinePlus