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Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function.
- Source :
-
RNA (New York, N.Y.) [RNA] 2012 Oct; Vol. 18 (10), pp. 1897-909. Date of Electronic Publication: 2012 Aug 23. - Publication Year :
- 2012
-
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.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution physiology
Catalytic Domain genetics
DNA Mutational Analysis
Genome, Protozoan
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Tertiary genetics
Protein Structure, Tertiary physiology
Protozoan Proteins chemistry
Protozoan Proteins genetics
Protozoan Proteins metabolism
RNA, Messenger analysis
RNA, Messenger genetics
Ribonucleoproteins chemistry
Ribonucleoproteins metabolism
Sequence Homology
Trypanosoma brucei brucei metabolism
RNA Editing genetics
RNA, Protozoan genetics
Ribonucleoproteins genetics
Ribonucleoproteins physiology
Trypanosoma brucei brucei genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1469-9001
- Volume :
- 18
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- RNA (New York, N.Y.)
- Publication Type :
- Academic Journal
- Accession number :
- 22919050
- Full Text :
- https://doi.org/10.1261/rna.035048.112