Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

Abstract

Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by ;20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 39-U-exonuclease [39-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 39-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.

The Architecture of Trypanosoma brucei editosomes

“…Each endonuclease also formed cross-links with KREPA6 and KREPB4 that similarly bridge the insertion and deletion subcomplexes, whereas KREN1 and KREN2 also cross-linked with KREPA3. RNAi knockdown of KREPA3 in PF results in partially disrupted editosomes that specifically lack endonuclease cleavage activity in vitro (40,64), supporting endonuclease proximity and interaction with KREPA3.…”

Endonuclease Associations with Three Distinct Editosomes in Trypanosoma brucei

“…The partial reduction of KREPA3 and KREPA6 in KREPB6 and KREPB7 subcomplexes isolated in the absence of endonuclease is consistent with the previous characterization of these proteins as a "bridge" between the deletion and insertion heterotrimeric subcomplexes, as the substoichiometric amount of these proteins may reflect the destabilizing effect caused by the absence of the insertion subcomplex. KREPA3 might directly interact with the endonucleases, as its presence has been shown to be crucial for cleavage activity (45). One striking difference between KREPB6, KREPB7, and KREPB8 complexes is the extent of reduction of KREPA6 in the absence of endonuclease expression (Fig.…”

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

“…Hence, KREPA3 may therefore have a role in binding the endonuclease component to the editosome. Interestingly, knockdown of KREPA3 expression in procyclic T. brucei did not affect U-deletion or U-insertion activities but led to complete loss of endonucleolytic activities that could be partially restored by adding recombinant KREPA3 to cell extracts (29,30). Thus, KREPA3 and KREPA6 together appear to provide key links for the integration of the three enzymatic subcomplexes responsible for endonucleolytic cleavage, U addition/ligation, and U removal/ligation in the core of the editosome.…”

“…It can directly interact with KREPA2 and KREPA6, and again this crucial structural role is consistent with functional studies. Knockdown of KREPA3 in procyclic form T. brucei resulted in accumulation of smaller complexes that appeared to be associations of the deletion and insertion subcomplexes with other proteins (30), and a more extensive knockdown in bloodstream forms also resulted in the virtual disappearance of ϳ20S editosomes (29). Furthermore, KREPA3 was shown here to also be able to directly interact with the RNase IIIlike protein KREPB5, which along with the related KREPB4 protein, has been suggested to function in association of the editosome endonucleases, KREN1, KREN2, and KREN3 (26).…”

“…3A, left panel, fractions 20 -25). The second peak, corresponding to an estimated molecular mass of 58 kDa, appeared to be a KREPA6 homotetramer (fractions [27][28][29][30]. Fractionation by size-exclusion chromatography of a mixture of individually purified KREPA6 and KREPA1 OB-fold domains (fragments 19 -148 and 626 -762*, respectively) suggested that these domains mediate the interaction: a shoulder migrating ahead of a major peak (the latter representing individual fragments) was only detectable in the presence of both fragments, indicative of formation of a heteromeric complex (supplemental Fig.…”