Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function

Abstract

The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.

The Architecture of Trypanosoma brucei editosomes

“…In contrast, the subcomplexes in KREN1-TAP eluates primarily contained insertion subcomplexes, as seen by the greater abundance of KREPA1 compared with KREPA2 and KREL1. These observations are consistent with previous studies that indicated that KREPB5 is most stably associated with the deletion subcomplex (30) and the endonucleases primarily associate with the insertion subcomplex (23,29). The compositions of the complexes and subcomplexes in the two different purifications provided a rationale for subsequent analysis of both KREN1-TAP and KREPB5-TAP eluates.…”

“…The proximity of KREPB4 to all endonucleases is intriguing given that KREPB4 is one of two editosome proteins, in addition to KREPB5, previously shown to contain a degenerate noncatalytic RNase III domain (30,31). We detected cross-links, and therefore proximity, between the RNase III domains of each of the endonucleases, and the region flanked by the U1-like zinc finger and RNase III domain, and the C-terminal domain in KREPB4 (Fig.…”

“…Biochemical and genetic experiments have predominantly revealed a network of protein-protein interactions that link two heterotrimeric subcomplexes within editosomes (23,29,30,(37)(38)(39)(45)(46)(47). However, the direct interactions of a variety of editosome proteins, including those that act at potentially key points of regulation during editing, were previously unidentified.…”

“…The common set of proteins contains two related proteins, KREPB4 and KREPB5, each of which has a U1-like zinc-finger motif, a Pumilio/fem-3 mRNA binding factor (PUF) motif, and a degenerate RNase III domain (30,31). Mutation of KREPB4 and KREPB5 residues that are universally conserved in all known catalytic RNase III domains has no effect on editing or in vitro cleavage of editing sites (30), whereas equivalent mutations in the RNase III domains of the KREN1, KREN2, or KREN3 endonucleases eliminate in vivo editing and in vitro cleavage of editing sites (22).…”

“…Mutation of KREPB4 and KREPB5 residues that are universally conserved in all known catalytic RNase III domains has no effect on editing or in vitro cleavage of editing sites (30), whereas equivalent mutations in the RNase III domains of the KREN1, KREN2, or KREN3 endonucleases eliminate in vivo editing and in vitro cleavage of editing sites (22). Because all characterized RNase IIIs function as dimers to cleave double-stranded RNA (dsRNA) (32,33), and because the endonucleases are each present as a single copy per editosome (23), we have therefore hypothesized that the noncatalytic RNase III domain of KREPB4 and KREPB5 forms a heterodimeric RNase III active site with the editing endonucleases.…”