Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

Lerch, Melissa; Carnes, Jason; Acestor, Nathalie; Guo, Xiaohong; Schnaufer, Achim; Stuart, Kenneth

doi:10.1128/ec.00046-12

Cited by 15 publications

(26 citation statements)

References 53 publications

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“…The only proteins known to directly associate with the z20S editosome that were absent from KREPB4 wild-type samples are those rarely detected in TAP-isolated complexes: KREH1, MEAT1, KREPB9, and KREPB10 (Panigrahi et al 2006;Aphasizheva et al 2009;Lerch et al 2012). Similar results were observed with wild-type KREPB5 samples, with KREPA5 additionally not detected.…”

Section: Analysis Of Editosomes Purified Via Tap-tagged Krepb4 and Krsupporting

confidence: 76%

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

Carnes¹,

Schnaufer²,

McDermott³

et al. 2012

RNA

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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.

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Section: Analysis Of Editosomes Purified Via Tap-tagged Krepb4 and Krsupporting

confidence: 76%

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

Carnes¹,

Schnaufer²,

McDermott³

et al. 2012

RNA

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“…Editosomes are ∼20S multiprotein complexes containing the enzymes that catalyze cycles of RNA editing, as well as proteins that have no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). 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).…”

Section: Discussionmentioning

confidence: 99%

“…The remaining KREPA family protein, KREPA5, was more recently identified as a common editosome subunit, and thus it was not included in previous analyses and has not yet been functionally characterized. A number of other proteins, KREPB9, KREPB10, and MEAT1, were also identified more recently and have been shown to copurify with the majority of proteins in ∼20 editosomes (27,44).…”

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confidence: 99%

“…Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

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confidence: 99%

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The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra-and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.cross-linking | proteomics | Trypanosoma brucei | RNA editing | editosome T he kinetoplastid Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in subSaharan Africa. Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA, which is composed of interlocked DNA maxicircles and minicircles (1, 2). Several identical maxicircles encode two ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing (3-9). RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).There are three similar versions of ∼20S editosomes that have a common set of 12 proteins (SI Appendix, Fig. S1 and Table S1). However, they are compositionally and functionally distinct in that each...

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“…The enzymes that catalyze RNA editing are in ϳ20S multiprotein editosome complexes that also contain proteins that have no known catalytic functions (14,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Three similar versions of these ϳ20S complexes exist, and all of them have a common set of 12 proteins, but each contains a different RNase III endonuclease and specific partner protein (25)(26)(27)(28)(29)32).…”

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Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

McDermott

Carnes

Stuart

2015

Molecular and Cellular Biology

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KREPB5 is an essential component of ϳ20S editosomes in Trypanosoma brucei which contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional null T. brucei bloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ϳ20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages of T. brucei that differentially edit mRNAs.T he kinetoplastid parasite Trypanosoma brucei is the etiologic agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in sub-Saharan Africa. T. brucei is a deeply divergent eukaryote, the study of which advanced the understanding of many fundamental biological processes and eukaryotic evolution. Indeed, a number of these processes, such as trans splicing, polycistronic transcription, antigenic variation, glycosylphosphatidylinositol anchoring, and mitochondrial RNA editing, were first described in trypanosomes and provided novel paradigms for eukaryotic biology (1-11). Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA (kDNA), within their single mitochondrion. The kDNA in T. brucei is comprised of ϳ50 identical maxicircles and thousands of heterogeneous minicircles (12, 13). The ϳ22-kb maxicircles encode two rRNAs and mRNAs for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing to generate translatable open reading frames (ORFs). RNA editing involves the precise insertion and deletion of uridylylates (Us) at hundreds and tens of editing sites (ESs), respectively. The minicircles encode numerous diverse ϳ60-nucleotide guide RNAs (gRNAs) which specify edited sequences (14-16). Editing progresses generally, but not precisely, 3= to 5= with respect to the mRNA, each gRNA specifies the editing of numerous ESs, and multiple gRNAs are required for complete editing of most transcripts. T. brucei undergoes stage-specific adaptations in the bloodstream of the mammalian host and in the tsetse fly, particularly in mitochondrial function (17,18), and a number of maxicircle transcripts are ...

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Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

Cited by 15 publications

References 53 publications

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

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

The Architecture of Trypanosoma brucei editosomes

Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

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