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

Abstract

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...

The Architecture of Trypanosoma brucei editosomes

“…Our CXMS data creates a detailed framework for the global architecture of editosome complexes, and can therefore provide context to reinterpret existing low-resolution electron microscopy data. For example, electron microscopy images of TAP-purified editosomes from T. brucei and Leishmania tarentolae revealed bipartite particles composed of two approximately equally sized, but structurally different globular domains that are connected via an interface (70,71). These studies also indicated that most editosome proteins are likely present as single copies in the complex, although the exact stoichiometry of editosomes remains unknown.…”

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

“…Tb REH1 was detected by mass spectrometry analysis in an MP63-immunoprecipitated sample and also in a RECC preparation isolated by ion-exchange chromatography (32)(33)(34). However, REH1 was not detected in REN1-, REN2-, or REN3-TAP pull-downs from T. brucei (32,35), nor in REL1-TAP and MP44-TAP pull-downs from L. tarentolae (7,11). These results could be explained by our finding that REH1 is associated with the RECC by RNA linkers, as are several other editingassociated complexes (6,11), and that this linkage is easily disrupted during the isolation.…”

“…The remaining proteins contain a variety of motifs including zinc fingers, single-strand binding (SSB), and OB folds, and the roles of these proteins in the editing reaction are not known, although several are required for structural stability of the RECC. Low-resolution cryoEM 3D structures for RECC particles from Trypanosoma brucei and Leishmania tarentolae have been published (7,10), but these do not have sufficient resolution to shed light on the precise mechanisms involved in the editing reactions.…”

“…The editing reaction is mediated by a holoenzyme or "editosome" (4). The core component of the editosome is the RECC (RNA editing core complex) (5) that contains approximately [15][16][17][18][19][20] polypeptides (2,(6)(7)(8)(9). Eight RECC proteins have been so far functionally characterized.…”

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

“…It remains to be investigated how the architecture of the core of the editosome described in this work can be integrated with the recently reported three-dimensional structures of 20S editosomes (55,56). Golas et al (56), using complexes purified via tagged KREPA3, reported a bipartite appearance in the majority of their structures, which might reflect the division into FIGURE 9.…”