Disruption of the Zinc Finger Motifs in the Leishmania tarentolae LC-4 (=TbMP63) L-complex Editing Protein Affects the Stability of the L-complex

Kang, Xuedong; Falick, Arnold M.; Nelson, Robert E.; Gao, Ge; Rogers, Kestrel; Aphasizhev, Ruslan; Simpson, Larry

doi:10.1074/jbc.m310185200

Cited by 23 publications

(23 citation statements)

References 21 publications

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“…2C). This confirms previous studies that showed their interaction in vivo and in vitro Kang et al 2004), and shows that the mutations did not affect their interaction. A small amount of the KREX2 proteins was nonspecifically immunoprecipitated, i.e., in the absence of KREPA2.…”

Section: Trypanosoma Brucei Krex1 and Krex2 Are Exouasessupporting

confidence: 81%

“…The difference between these results probably reflects the larger amounts of the enzymes used in the previous studies than with the in vitro transcribed/translated proteins described here, although differences in the dsRNA substrate and/or assay conditions cannot be excluded. Nevertheless, interaction of KREX2 with its binding partner KREPA2 Kang et al 2004) resulted in efficient removal of Us from both ssRNA and the dsRNA ( Fig. 2; data not shown).…”

Section: Discussionmentioning

confidence: 99%

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Differential functions of two editosome exoUases in Trypanosoma brucei

Ernst

Panicucci

Carnes

et al. 2009

RNA

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Mitochondrial RNAs in trypanosomes are edited by the insertion and deletion of uridine (U) nucleotides to form translatable mRNAs. Editing is catalyzed by three distinct editosomes that contain two related U-specific exonucleases (exoUases), KREX1 and KREX2, with the former present exclusively in KREN1 editosomes and the latter present in all editosomes. We show here that repression of KREX1 expression leads to a concomitant reduction of KREN1 in ;20S editosomes, whereas KREX2 repression results in reductions of KREPA2 and KREL1 in ;20S editosomes. Knockdown of KREX1 results in reduced cell viability, reduction of some edited RNA in vivo, and a significant reduction in deletion but not insertion endonuclease activity in vitro. In contrast, KREX2 knockdown does not affect cell growth or editing in vivo but results in modest reductions of both insertion and deletion endonuclease activities and a significant reduction of U removal in vitro. Simultaneous knockdown of both proteins leads to a more severe inhibition of cell growth and editing in vivo and an additive effect on endonuclease cleavage in vitro. Taken together, these results indicate that both KREX1 and KREX2 are important for retention of other proteins in editosomes, and suggest that the reduction in cell viability upon KREX1 knockdown is likely a consequence of KREN1 loss. Furthermore, although KREX2 appears dispensable for cell growth, the increased inhibition of editing and parasite viability upon knockdown of both KREX1 and KREX2 together suggests that both proteins have roles in editing.

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Section: Trypanosoma Brucei Krex1 and Krex2 Are Exouasessupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Differential functions of two editosome exoUases in Trypanosoma brucei

Ernst

Panicucci

Carnes

et al. 2009

RNA

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“…The REL1-TAP pull-down showed a minor amount of the endogenous REL1 in addition to the tagged REL1 (14). Similar results were obtained for another component of the REL1 subcomplex-the LC4 (MP63) protein (17). These results suggest that there is a minor percentage of the REL1 subcomplexes that are dimeric, but the majority are single-copy.…”

supporting

confidence: 73%

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

Li¹,

Hui

et al. 2009

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

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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...

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“…This is consistent with coimmunoprecipitation experiments using recombinant protein, which showed that mutating the KREPA2 zinc fingers did not affect pulldown of KREL1 (data not shown). Interestingly, expression of LtKREPA2 with disrupted zinc fingers in Leishmania resulted in substantial breakdown of the 20S editosome (54), suggesting that these motifs do have a functional role, possibly in interaction with KREX2 or perhaps in proper folding of KREPA2 itself. The latter would be consistent with the finding that correct folding of recombinant KREPA3, which also contains two zinc finger motifs, required zinc ions (47).…”

Section: Discussionmentioning

confidence: 99%

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

Schnaufer

Park

et al. 2010

Journal of Biological Chemistry

126

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Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ϳ20S on glycerol gradients. These ϳ20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ϳ20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ϳ20S editosomes during the editing process.

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Disruption of the Zinc Finger Motifs in the Leishmania tarentolae LC-4 (=TbMP63) L-complex Editing Protein Affects the Stability of the L-complex

Cited by 23 publications

References 21 publications

Differential functions of two editosome exoUases in Trypanosoma brucei

Differential functions of two editosome exoUases in Trypanosoma brucei

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

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

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