Kinetoplastid RNA editing involves a 3′ nucleotidyl phosphatase activity

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.

Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

“…Alternatively, they could result from cleavage 3= rather than 5= to the phosphate, although this is unprecedented among RNase IIIs. However, editosome-associated 3= phosphatase activity has been reported (33). Overall, these results suggest that KREPB9 and KREPB10 function may expand the endonuclease repertoire for recognition and cleavage of the hundreds of editing sites that are generally similar partial RNA duplex substrates but have distinct sequences and structures.…”

RNA-level unscrambling of fragmented genes inDiplonemamitochondria

“…In turn, TbMP99 and TbMP100 exhibit 3'-specific nucleotidyl phosphatase activity converting the 3'NMP to a 3' hydroxyl group, which permits pre-mRNA re-ligation. 35 This dephosphorylation step was proposed to serve as quality control in trypanosome RNA editing: only when the number of inserted nucleotides permits full pairing with the gRNA, the 3'monophosphate would be removed from the terminal U, and re-sealing of pre-mRNA would proceed. 35 Kinetoplastid editosomes include virtually all catalytic activities required for post-transcriptional processes in Diplonema mitochondria, notably an endoribonuclease for module end processing, a TUTase for U addition, an exoUase leaving the 3'NMP, a 3'-nucleotidyl phosphatase that "repairs" 3' termini generated by this exonuclease and, finally, RNA ligase for module joining.…”

“…35 This dephosphorylation step was proposed to serve as quality control in trypanosome RNA editing: only when the number of inserted nucleotides permits full pairing with the gRNA, the 3'monophosphate would be removed from the terminal U, and re-sealing of pre-mRNA would proceed. 35 Kinetoplastid editosomes include virtually all catalytic activities required for post-transcriptional processes in Diplonema mitochondria, notably an endoribonuclease for module end processing, a TUTase for U addition, an exoUase leaving the 3'NMP, a 3'-nucleotidyl phosphatase that "repairs" 3' termini generated by this exonuclease and, finally, RNA ligase for module joining. This raises the question whether in Diplonema mitochondria these activities are exerted by homologs of the kinetoplastid enzymes and whether the enzymes are also organized in a multifunctional protein complex.…”

Structure and intrinsic disorder of the proteins of theTrypanosoma brucei editosome

“…These diverse enzymes share a common catalytic mechanism of cleaving phosphodiester bonds. The domain is present in the C-terminal regions of TbMP99 and TbMP100 and both proteins have been shown to execute 3 0 nucleotidyl phosphatase activity in vitro [61].…”