Novel TUTase associates with an editosome-like complex in mitochondria of <i>Trypanosoma brucei</i>

Aphasizheva, Inna; Ringpis, Gene-Errol; Weng, James; Gershon, Paul D.; Lathrop, Richard H.; Aphasizhev, Ruslan

doi:10.1261/rna.1538809

Cited by 29 publications

(37 citation statements)

References 59 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: 72%

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: 72%

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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“…Less protein was recovered in the calmodulin binding protein eluates (CBE), but the relative proportions of the proteins were similar to those of the TEV eluates. In addition, Western analysis with a polyclonal antibody to the mitochondrial editosome-like complex-associated TUTase (MEAT1) revealed the presence of this protein in the purified KREPB9, KREPB10, and KREPB5 complexes (5). SYPRO rubystained SDS-PAGE gels of the calmodulin column eluates revealed profiles that were similar to each other's and to those of editing complexes, albeit with some differences in the protein ratios.…”

Section: Identification Of Krepb9 and Krepb10 Krepb9 And Krepb10mentioning

confidence: 96%

“…Other complexes and proteins participate in RNA editing and/or other steps in mitochondrial RNA processing (1,3,16,17,21,50). The Mitochondrial Editosome-like complex-Associated TUTase 1 (MEAT1) has been shown to associate with editosome proteins, and this protein adds uridines to RNA substrates in vitro (5).…”

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confidence: 99%

Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

et al. 2012

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bMultiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ϳ20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5= OH on the 3= product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei.

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“…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: 96%

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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Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei

Cited by 29 publications

References 59 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

Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

The Architecture of Trypanosoma brucei editosomes

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