Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function.
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.
Seattle Biomedical Research Institute, Seattle, Washington 98109, USA.
Jason
J
Carnes
Achim
A
Schnaufer
Suzanne M
SM
McDermott
Gonzalo
G
Domingo
Rose
R
Proff
Alodie G
AG
Steinberg
Irina
I
Kurtz
Kenneth
K
Stuart
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D020134:Catalytic Domain / Q000235:genetics; D004252:DNA Mutational Analysis; D018503:Genome, Protozoan; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D017385:Sequence Homology; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012261:Ribonucleoproteins