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Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

Niemann M, Kaibel H, Schlüter E, Weitzel K, Brecht M, Göringer HU - Nucleic Acids Res. (2009)

Bottom Line: The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity.However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity.Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

View Article: PubMed Central - PubMed

Affiliation: Genetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

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Related in: MedlinePlus

Comparison of exoUase reaction products derived from incubations with r42 and 20S editosomes. (A) Sketch of the input precleaved deletion mRNA/gRNA hybrid molecule (5′ cleavage fragment (CF), black, 3′ CF, grey). An asterisk designates the radioactive phosphate group. OH and NH2 indicate 3′ terminal hydroxyl or amino groups. (B) Electrophoretic analysis of the reaction products of the pre-mRNA/gRNA hybrid shown in (A) after incubation with r42 or 20S editosomes. A schematic representation of the processed RNAs is given on the right. Mock represents an RNA sample incubated in the absence of 20S editosomes or r42.
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Figure 2: Comparison of exoUase reaction products derived from incubations with r42 and 20S editosomes. (A) Sketch of the input precleaved deletion mRNA/gRNA hybrid molecule (5′ cleavage fragment (CF), black, 3′ CF, grey). An asterisk designates the radioactive phosphate group. OH and NH2 indicate 3′ terminal hydroxyl or amino groups. (B) Electrophoretic analysis of the reaction products of the pre-mRNA/gRNA hybrid shown in (A) after incubation with r42 or 20S editosomes. A schematic representation of the processed RNAs is given on the right. Mock represents an RNA sample incubated in the absence of 20S editosomes or r42.

Mentions: The described result raises the question whether the Np-generating specificity of r42 is relevant within the context of the RNA-editing reaction cycle. For that we compared the r42-derived exonucleolytic reaction products with RNA fragments generated by incubation with 20S editosomes. Using the precleaved U deletion in vitro editing system developed by Seiwert and Stuart (41), we monitored the gRNA-directed exonucleolytic degradation of 4 Us from a radiolabeled 5′ pre-mRNA fragment. The generated products were separated in denaturing, high-resolution polyacrylamide gels, in which the presence or absence of a terminal phosphate group can be visualized by an ∼0.5 nt offset (53). Figure 2 shows a typical experiment. r42 degrades the ss U-overhang of the input 5′ pre-mRNA fragment in a distributive fashion and the generated RNAs have an identical electrophoretic mobility as fragments generated by incubation with 20S editosomes. This indicates that the fragments not only have the same nt length but also the same phosphorylation status. Thus, the exoUase activities of 20S editosomes and of r42 generate 3′ phosphate termini.Figure 2.


Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

Niemann M, Kaibel H, Schlüter E, Weitzel K, Brecht M, Göringer HU - Nucleic Acids Res. (2009)

Comparison of exoUase reaction products derived from incubations with r42 and 20S editosomes. (A) Sketch of the input precleaved deletion mRNA/gRNA hybrid molecule (5′ cleavage fragment (CF), black, 3′ CF, grey). An asterisk designates the radioactive phosphate group. OH and NH2 indicate 3′ terminal hydroxyl or amino groups. (B) Electrophoretic analysis of the reaction products of the pre-mRNA/gRNA hybrid shown in (A) after incubation with r42 or 20S editosomes. A schematic representation of the processed RNAs is given on the right. Mock represents an RNA sample incubated in the absence of 20S editosomes or r42.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665232&req=5

Figure 2: Comparison of exoUase reaction products derived from incubations with r42 and 20S editosomes. (A) Sketch of the input precleaved deletion mRNA/gRNA hybrid molecule (5′ cleavage fragment (CF), black, 3′ CF, grey). An asterisk designates the radioactive phosphate group. OH and NH2 indicate 3′ terminal hydroxyl or amino groups. (B) Electrophoretic analysis of the reaction products of the pre-mRNA/gRNA hybrid shown in (A) after incubation with r42 or 20S editosomes. A schematic representation of the processed RNAs is given on the right. Mock represents an RNA sample incubated in the absence of 20S editosomes or r42.
Mentions: The described result raises the question whether the Np-generating specificity of r42 is relevant within the context of the RNA-editing reaction cycle. For that we compared the r42-derived exonucleolytic reaction products with RNA fragments generated by incubation with 20S editosomes. Using the precleaved U deletion in vitro editing system developed by Seiwert and Stuart (41), we monitored the gRNA-directed exonucleolytic degradation of 4 Us from a radiolabeled 5′ pre-mRNA fragment. The generated products were separated in denaturing, high-resolution polyacrylamide gels, in which the presence or absence of a terminal phosphate group can be visualized by an ∼0.5 nt offset (53). Figure 2 shows a typical experiment. r42 degrades the ss U-overhang of the input 5′ pre-mRNA fragment in a distributive fashion and the generated RNAs have an identical electrophoretic mobility as fragments generated by incubation with 20S editosomes. This indicates that the fragments not only have the same nt length but also the same phosphorylation status. Thus, the exoUase activities of 20S editosomes and of r42 generate 3′ phosphate termini.Figure 2.

Bottom Line: The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity.However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity.Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

View Article: PubMed Central - PubMed

Affiliation: Genetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

Show MeSH
Related in: MedlinePlus