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Trypanosome MTR4 is involved in rRNA processing.

Cristodero M, Clayton CE - Nucleic Acids Res. (2007)

Bottom Line: The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the 'TRAMP' complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p.Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect.These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the 'TRAMP' complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.

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Depletion of TbMTR4 by inducible RNAi affects cell growth. (A) Trypanosomes expressing a TbMTR4-specfic dsRNA were grown in the absence (solid line) and presence (dashed line) of 100 ng/ml tetracycline to induce RNAi-mediated depletion. Cultures were followed for 3 days and diluted to 0.5 × 106 cells/ml when required. The inset is a northern blot showing depletion of the MTR4 mRNA; rRNA staining was similar in all lanes (data not shown). (B) Effect of depletion of TbMTR4 on 5.8S rRNA maturation in vivo. Total RNA was extracted from trypanosomes grown in the absence (−) or presence (+) of tetracycline for 24 h, and separated in polyacrylamide–urea gels. The gels were transferred to nylon membranes and hybridized with probes to detect extended or mature 5.8S rRNA. Mature 5.8S and full-length 7S rRNA are shown, while the incompletely processed species are marked with an asterisk. Cells depleted of the exosome subunit RRP6 were used as the control for incomplete rRNA processing.
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Figure 2: Depletion of TbMTR4 by inducible RNAi affects cell growth. (A) Trypanosomes expressing a TbMTR4-specfic dsRNA were grown in the absence (solid line) and presence (dashed line) of 100 ng/ml tetracycline to induce RNAi-mediated depletion. Cultures were followed for 3 days and diluted to 0.5 × 106 cells/ml when required. The inset is a northern blot showing depletion of the MTR4 mRNA; rRNA staining was similar in all lanes (data not shown). (B) Effect of depletion of TbMTR4 on 5.8S rRNA maturation in vivo. Total RNA was extracted from trypanosomes grown in the absence (−) or presence (+) of tetracycline for 24 h, and separated in polyacrylamide–urea gels. The gels were transferred to nylon membranes and hybridized with probes to detect extended or mature 5.8S rRNA. Mature 5.8S and full-length 7S rRNA are shown, while the incompletely processed species are marked with an asterisk. Cells depleted of the exosome subunit RRP6 were used as the control for incomplete rRNA processing.

Mentions: One function of yeast Mtr4p is the stimulation of the nuclear exosome. We first wanted to find out whether trypanosome MTR4 has a similar function. Depletion of TbMTR4 by RNAi caused growth arrest within 48 h, followed by death (Figure 2A). [Addition of tetracycline to trypanosomes without inducible constructs does not affect growth, the transcriptome or the proteome (34).] We previously showed that in trypanosomes, the nuclear exosome is involved in the processing of 7S to the mature 5.8S rRNA (24). Analysis of rRNA 24 h after depletion of TbMTR4 showed that there was a 2-fold increase in the ratio of 7S to 5.8S; for the exosome subunit TbRRP6 the increase was more than 7-fold (Figure 2B).Figure 2.


Trypanosome MTR4 is involved in rRNA processing.

Cristodero M, Clayton CE - Nucleic Acids Res. (2007)

Depletion of TbMTR4 by inducible RNAi affects cell growth. (A) Trypanosomes expressing a TbMTR4-specfic dsRNA were grown in the absence (solid line) and presence (dashed line) of 100 ng/ml tetracycline to induce RNAi-mediated depletion. Cultures were followed for 3 days and diluted to 0.5 × 106 cells/ml when required. The inset is a northern blot showing depletion of the MTR4 mRNA; rRNA staining was similar in all lanes (data not shown). (B) Effect of depletion of TbMTR4 on 5.8S rRNA maturation in vivo. Total RNA was extracted from trypanosomes grown in the absence (−) or presence (+) of tetracycline for 24 h, and separated in polyacrylamide–urea gels. The gels were transferred to nylon membranes and hybridized with probes to detect extended or mature 5.8S rRNA. Mature 5.8S and full-length 7S rRNA are shown, while the incompletely processed species are marked with an asterisk. Cells depleted of the exosome subunit RRP6 were used as the control for incomplete rRNA processing.
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Figure 2: Depletion of TbMTR4 by inducible RNAi affects cell growth. (A) Trypanosomes expressing a TbMTR4-specfic dsRNA were grown in the absence (solid line) and presence (dashed line) of 100 ng/ml tetracycline to induce RNAi-mediated depletion. Cultures were followed for 3 days and diluted to 0.5 × 106 cells/ml when required. The inset is a northern blot showing depletion of the MTR4 mRNA; rRNA staining was similar in all lanes (data not shown). (B) Effect of depletion of TbMTR4 on 5.8S rRNA maturation in vivo. Total RNA was extracted from trypanosomes grown in the absence (−) or presence (+) of tetracycline for 24 h, and separated in polyacrylamide–urea gels. The gels were transferred to nylon membranes and hybridized with probes to detect extended or mature 5.8S rRNA. Mature 5.8S and full-length 7S rRNA are shown, while the incompletely processed species are marked with an asterisk. Cells depleted of the exosome subunit RRP6 were used as the control for incomplete rRNA processing.
Mentions: One function of yeast Mtr4p is the stimulation of the nuclear exosome. We first wanted to find out whether trypanosome MTR4 has a similar function. Depletion of TbMTR4 by RNAi caused growth arrest within 48 h, followed by death (Figure 2A). [Addition of tetracycline to trypanosomes without inducible constructs does not affect growth, the transcriptome or the proteome (34).] We previously showed that in trypanosomes, the nuclear exosome is involved in the processing of 7S to the mature 5.8S rRNA (24). Analysis of rRNA 24 h after depletion of TbMTR4 showed that there was a 2-fold increase in the ratio of 7S to 5.8S; for the exosome subunit TbRRP6 the increase was more than 7-fold (Figure 2B).Figure 2.

Bottom Line: The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the 'TRAMP' complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p.Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect.These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the 'TRAMP' complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.

Show MeSH
Related in: MedlinePlus