TbMP42, a Protein Component of the RNA Editing Complex in African Trypanosomes, Has Endo-Exoribonuclease Activity

Abstract

RNA editing in trypanosomatids is catalyzed by a high molecular mass RNP complex, which is only partially characterized. TbMP42 is a 42 kDa protein of unknown function that copurifies with the editing complex. The polypeptide is characterized by two Zn fingers and a potential barrel structure/OB-fold at its C terminus. Using recombinant TbMP42, we show that the protein can bind to dsRNA and dsDNA but fails to recognize DNA/RNA hybrids. rTbMP42 degrades ssRNA by a 3' to 5' exoribonuclease activity. In addition, rTbMP42 has endoribonuclease activity, which preferentially hydrolyzes non-base-paired uridylate-containing sequences. Gene silencing of TbMP42 inhibits cell growth and is ultimately lethal to the parasite. Mitochondrial extracts from TbMP42-minus trypanosomes have only residual RNA editing activity and strongly reduced endo-exoribonuclease activity. However, all three activities can be restored by the addition of rTbMP42. Together, the data suggest that TbMP42 contributes both endo- and exoribonuclease activity to the editing reaction cycle.

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

“…Interestingly, expression of LtKREPA2 with disrupted zinc fingers in Leishmania resulted in substantial breakdown of the 20S editosome (54), suggesting that these motifs do have a functional role, possibly in interaction with KREX2 or perhaps in proper folding of KREPA2 itself. The latter would be consistent with the finding that correct folding of recombinant KREPA3, which also contains two zinc finger motifs, required zinc ions (47). Expression of KREPA3 with mutated zinc fingers in T. brucei still resulted in incorporation into editosomes but failed to rescue the lethal effect of depleting endogenous KREPA3 (29).…”

“…Eukaryotic SSBs typically form hetero-oligomers with multiple OB-fold domains in which some OB-folds are responsible for nucleic acid binding while others mediate protein-protein interactions, often with multiple partners (45,46). Recombinant KREPA6, KREPA3, and KREPA4 all have been shown to be able to bind RNA in vitro, with a preference for gRNA and oligo(U) sequences (32,34,47). The RNA-binding activity of KREPA3 was mapped to the OB-fold domain (47).…”

“…Recombinant KREPA6, KREPA3, and KREPA4 all have been shown to be able to bind RNA in vitro, with a preference for gRNA and oligo(U) sequences (32,34,47). The RNA-binding activity of KREPA3 was mapped to the OB-fold domain (47). In addition, there is evidence that the OB-fold domains of KREPA1 and KREPA2 provide substrate-binding platforms for the enzymatic activities in the deletion and insertion subcomplexes, respectively (25,48).…”

RNA-level unscrambling of fragmented genes inDiplonemamitochondria

“…Functional characterization of mitochondrial proteins in trypanosomes revealed three enzymes implicated in U-insertion/deletion RNA editing, TbMP42, TbMP99 and TbMP100. 33,34 TbMP42 displays in vitro exoUase activity leaving 3'-monophosphate ends, which, as in Diplonema, cannot be ligated with the 5' terminus of the downstream pre-mRNA cleavage fragment. 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.…”

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

“…Although TbMP90 and TbMP61 were stated in recent reviews (1,16) to also contain double-strand RNA-binding motifs, such motifs are not readily identifiable (L.S., unpublished data). In addition, the MP42 L-complex protein, which has only zinc finger (ZnF C2H2) and single-strand RNA-binding (SSB) motifs, nevertheless exhibited both exonuclease and endonuclease activities, but the activities identified did not show the required specificity, and the role of this protein remains an open question (17).…”