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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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A polyadenylated rRNA precursor accumulates in TbMTR4 RNAi cells. (A) Schematic diagram of the rRNA transcription unit and processing in T. brucei. (B) Primers used for RT-PCR analysis. (C) Total RNA was extracted from trypanosomes growing in the absence (−) or presence (+) of tetracycline and reverse transcribed using the anchored oligo(dT) primer. The cDNA was PCR amplified (25 cycles) with primers specific for ITS7 and SR6 rRNA, and the products separated on a 2% agarose gel. For lanes 1, 3, 5, 7 and 9, undiluted cDNA was used; lanes 2, 4, 6, 8 and 10 are 10-fold dilutions. Quantitation of the 10× lanes revealed that the TbNPAPL line had a 30% reduction in signal in the absence of tetracycline (lanes 7 and 8) and a 50% reduction in the presence of tetracycline (lanes 9 and 10). (D) The relative signals for ITS7 were quantified by scanning across the gel. The wild-type pattern (lane 1) is shown as a solid line, the TbMTR4-depleted pattern (lane 5) by a dashed line and the TbNPAPL-depleted pattern (lane 9) by the gray solid line. When the TbNPAPL profile was stretched vertically in the graphics program, the overall pattern was indistinguishable from wild type.
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Figure 4: A polyadenylated rRNA precursor accumulates in TbMTR4 RNAi cells. (A) Schematic diagram of the rRNA transcription unit and processing in T. brucei. (B) Primers used for RT-PCR analysis. (C) Total RNA was extracted from trypanosomes growing in the absence (−) or presence (+) of tetracycline and reverse transcribed using the anchored oligo(dT) primer. The cDNA was PCR amplified (25 cycles) with primers specific for ITS7 and SR6 rRNA, and the products separated on a 2% agarose gel. For lanes 1, 3, 5, 7 and 9, undiluted cDNA was used; lanes 2, 4, 6, 8 and 10 are 10-fold dilutions. Quantitation of the 10× lanes revealed that the TbNPAPL line had a 30% reduction in signal in the absence of tetracycline (lanes 7 and 8) and a 50% reduction in the presence of tetracycline (lanes 9 and 10). (D) The relative signals for ITS7 were quantified by scanning across the gel. The wild-type pattern (lane 1) is shown as a solid line, the TbMTR4-depleted pattern (lane 5) by a dashed line and the TbNPAPL-depleted pattern (lane 9) by the gray solid line. When the TbNPAPL profile was stretched vertically in the graphics program, the overall pattern was indistinguishable from wild type.

Mentions: In trypanosomes, the rRNA is transcribed as a pre-RNA which is processed to the individual mature species by endonucleolytic cleavage and 3′ trimming (Figure 4A). Unusually, the large subunit RNA is cut into several smaller pieces, which are subsequently trimmed (35). Polyadenylated Leishmania rRNAs corresponding to SR6 rRNA and a partially processed precursor including the ITS7 were previously reported (27). To look for similar polyadenylated rRNAs in T. brucei, we made cDNA by priming with oligo d(T) bearing a 5′ anchor sequence, then amplified the polyadenylated rRNA species using specific primers (Figure 4B). Both polyadenylated SR6 rRNA and the longer polyadenylated ITS7 intermediate (Figure 4C, lanes 1 and 2) could be detected in trypanosomes. To quantitate the results from different cell lines, we compared the amounts of PCR product—including both the main band and longer species—in serial 10-fold dilutions of the cDNA, using polyadenylated tubulin mRNA as the standard (Figure 4C).Figure 4.


Trypanosome MTR4 is involved in rRNA processing.

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

A polyadenylated rRNA precursor accumulates in TbMTR4 RNAi cells. (A) Schematic diagram of the rRNA transcription unit and processing in T. brucei. (B) Primers used for RT-PCR analysis. (C) Total RNA was extracted from trypanosomes growing in the absence (−) or presence (+) of tetracycline and reverse transcribed using the anchored oligo(dT) primer. The cDNA was PCR amplified (25 cycles) with primers specific for ITS7 and SR6 rRNA, and the products separated on a 2% agarose gel. For lanes 1, 3, 5, 7 and 9, undiluted cDNA was used; lanes 2, 4, 6, 8 and 10 are 10-fold dilutions. Quantitation of the 10× lanes revealed that the TbNPAPL line had a 30% reduction in signal in the absence of tetracycline (lanes 7 and 8) and a 50% reduction in the presence of tetracycline (lanes 9 and 10). (D) The relative signals for ITS7 were quantified by scanning across the gel. The wild-type pattern (lane 1) is shown as a solid line, the TbMTR4-depleted pattern (lane 5) by a dashed line and the TbNPAPL-depleted pattern (lane 9) by the gray solid line. When the TbNPAPL profile was stretched vertically in the graphics program, the overall pattern was indistinguishable from wild type.
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Figure 4: A polyadenylated rRNA precursor accumulates in TbMTR4 RNAi cells. (A) Schematic diagram of the rRNA transcription unit and processing in T. brucei. (B) Primers used for RT-PCR analysis. (C) Total RNA was extracted from trypanosomes growing in the absence (−) or presence (+) of tetracycline and reverse transcribed using the anchored oligo(dT) primer. The cDNA was PCR amplified (25 cycles) with primers specific for ITS7 and SR6 rRNA, and the products separated on a 2% agarose gel. For lanes 1, 3, 5, 7 and 9, undiluted cDNA was used; lanes 2, 4, 6, 8 and 10 are 10-fold dilutions. Quantitation of the 10× lanes revealed that the TbNPAPL line had a 30% reduction in signal in the absence of tetracycline (lanes 7 and 8) and a 50% reduction in the presence of tetracycline (lanes 9 and 10). (D) The relative signals for ITS7 were quantified by scanning across the gel. The wild-type pattern (lane 1) is shown as a solid line, the TbMTR4-depleted pattern (lane 5) by a dashed line and the TbNPAPL-depleted pattern (lane 9) by the gray solid line. When the TbNPAPL profile was stretched vertically in the graphics program, the overall pattern was indistinguishable from wild type.
Mentions: In trypanosomes, the rRNA is transcribed as a pre-RNA which is processed to the individual mature species by endonucleolytic cleavage and 3′ trimming (Figure 4A). Unusually, the large subunit RNA is cut into several smaller pieces, which are subsequently trimmed (35). Polyadenylated Leishmania rRNAs corresponding to SR6 rRNA and a partially processed precursor including the ITS7 were previously reported (27). To look for similar polyadenylated rRNAs in T. brucei, we made cDNA by priming with oligo d(T) bearing a 5′ anchor sequence, then amplified the polyadenylated rRNA species using specific primers (Figure 4B). Both polyadenylated SR6 rRNA and the longer polyadenylated ITS7 intermediate (Figure 4C, lanes 1 and 2) could be detected in trypanosomes. To quantitate the results from different cell lines, we compared the amounts of PCR product—including both the main band and longer species—in serial 10-fold dilutions of the cDNA, using polyadenylated tubulin mRNA as the standard (Figure 4C).Figure 4.

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