Mass spectrometric analysis of the editosome and other multiprotein complexes in Trypanosoma brucei

Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing "block." Many mRNAs require multiple gRNAs; the observed overall 3′ to 5′ polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. Hel61, a mitochondrial DEAD-box protein, was previously shown to be involved in RNA editing, but the functional role was not clear. Here we report that down-regulation of Hel61 [renamed REH1 (RNA editing helicase 1)] expression in Trypanosoma brucei selectively affects editing mediated by two or more overlapping gRNAs but has no effect on editing within a single block. Down-regulation produces an increased abundance of the gRNA/edited mRNA duplex for the first editing block of the A6 mRNA. Recombinant REH1 has an ATP-dependent double strand RNA unwinding activity in vitro with a model gRNA-mRNA duplex. These data indicate that REH1 is involved in gRNA displacement either directly by unwinding the gRNA/edited mRNA duplex or indirectly, to allow the 5′ adjacent upstream gRNA to form an anchor duplex with the edited mRNA to initiate another block of editing. Purified tagged REH1 is associated with the RNA editing core complex by RNA linkers and a colocalization of REH1, REL1, and two kinetoplast ribosomal proteins with the kinetoplast DNA was observed by immunofluorescence, suggesting that editing, transcription, and translation may be functionally linked.Leishmania | RNAi | tandem affinity purification | streptavidin binding and protein A purification

Section: Discussionmentioning

confidence: 93%

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

Herrera

Zhou

et al. 2011

“…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).…”

mentioning

confidence: 99%

The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

Self Cite

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...

“…We previously proposed a model for the mechanism of this editing process involving a cleavage, addition or deletion of U's and ligation progressing in a 3Ј to 5Ј polarity (4), and this model has been since experimentally validated in almost every detail (5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria

Li¹,

Hui

et al. 2009

Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or ''L (ligase)-complex'' from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20 -25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another singleparticle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction.editing ͉ electron microscopy ͉ kinetoplast ͉ Leishmania ͉ trypanosome U ridine (U) insertion/deletion RNA editing is a posttranscriptional process in mitochondria of kinetoplastid protists that involves the modification of mRNA transcripts of mitochondrial cryptogenes by precise insertion and deletion of U residues to create translatable sequences (1-3). We previously proposed a model for the mechanism of this editing process involving a cleavage, addition or deletion of U's and ligation progressing in a 3Ј to 5Ј polarity (4), and this model has been since experimentally validated in almost every detail (5-10).Editing involves multiple multiprotein complexes associated by RNA linkers. The core editing complex, known as the L-complex, ''20S complex,'' ''editosome,'' or ''ϳ20S editosome,'' was detected by following the sedimentation and gel filtration of 2 adenylatable RNA ligases using Leishmania tarentolae and Trypanosoma brucei mitochondrial extracts (11,12). The activity sedimented around 20 -25S and migrated as a single band in a native gel (13). A ϳ20S multiprotein complex that showed both U-insertion and U-deletion in vitro activities was also purified from T. brucei mitochondria by column chromatography. An improved isolation of the L-complex from L. tarentolae was achieved by the tandem affinity purification procedure (TA P) (14); TA P-tagged plasmidexpressed REL1 became incorporated into the L-complex in vivo, allowing isolation of tagged complexes.Fourteen proteins were initially identified by mass spectrometry of the L. tarentolae REL1-TAP pull-down (14). All of the L. tarentolae proteins had homologs in T. brucei, and several additional proteins found in the T. brucei L-complex (15) were later identified in L. tarentolae (Tables S1 and S2).Two 3-prot...