TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

Hashimi, Hassan; Zı́ková, Alena; Panigrahi, Aswini K.; Stuart, Kenneth; Lukeš, Julius

doi:10.1261/rna.888808

Cited by 84 publications

(150 citation statements)

References 38 publications

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“…Changes in unedited mRNAs were not statistically significant, as in other studies showing sublethal editing effects, including with our REH2 construct above, where unedited RNAs are not always evidently affected (Supplemental Fig. S4; Hashimi et al 2008;Acestor et al 2009). The never-edited mRNA COI was not affected, while the A6 mRNA may only be slightly decreased.…”

Section: Resultssupporting

confidence: 50%

“…Inducible knockdown of mRPN1 results in the loss of gRNAs and accumulation of precursor sequences (pre-gRNAs), consistent with a role in processing. Finally, cell purified mRPN1 has nuclease-resistant interactions with three other proteins that were previously identified in relatively large MRB (mitochondrial RNA binding)-related complexes, which do not have mRPN1, and appear to have multiple roles in mitochondrial RNA metabolism (Hashimi et al 2008;Panigrahi et al 2008;Weng et al 2008;Hernandez et al 2010;Ammerman et al 2011). One of these factors (TbRGG2) directly binds mRPN1 and was previously linked with gRNA utilization during editing, which may include entry into the editing pathway.…”

Section: Introductionmentioning

confidence: 99%

“…Some factors affect editing indirectly, for example, by impacting the level of pre-mRNA precursors or the turnover of gRNA or mRNA in vivo, or possibly through associated activities identified in vitro: RNA annealing, unwinding, or 39 remodeling (Aphasizhev et al 2002;Pelletier and Read 2003;Mingler et al 2006;Etheridge et al 2008;Fisk et al 2008;Hashimi et al 2008;Panigrahi et al 2008;Weng et al 2008;Koslowsky 2009;Aphasizheva and Aphasizhev 2010;Hernandez et al 2010). Finally, a mitochondrial activity was found to catalyze 39 processing of polycistronic pre-gRNAs, but the responsible nucleases are not known (Grams et al 2000).…”

Section: Introductionmentioning

confidence: 99%

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Guide RNA biogenesis involves a novel RNase III family endoribonuclease inTrypanosoma brucei

Madina¹,

Gokulan²,

Vashisht³

et al. 2011

RNA

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The mitochondrial genome of kinetoplastids, including species of Trypanosoma and Leishmania, is an unprecedented DNA structure of catenated maxicircles and minicircles. Maxicircles represent the typical mitochondrial genome encoding components of the respiratory complexes and ribosomes. However, most mRNA sequences are cryptic, and their maturation requires a unique U insertion/deletion RNA editing. Minicircles encode hundreds of small guide RNAs (gRNAs) that partially anneal with unedited mRNAs and direct the extensive editing. Trypanosoma brucei gRNAs and mRNAs are transcribed as polycistronic precursors, which undergo processing preceding editing; however, the relevant nucleases are unknown. We report the identification and functional characterization of a close homolog of editing endonucleases, mRPN1 (mitochondrial RNA precursor-processing endonuclease 1), which is involved in gRNA biogenesis. Recombinant mRPN1 is a dimeric dsRNAdependent endonuclease that requires Mg 2+ , a critical catalytic carboxylate, and generates 2-nucleotide 39 overhangs. The cleavage specificity of mRPN1 is reminiscent of bacterial RNase III and thus is fundamentally distinct from editing endonucleases, which target a single scissile bond just 59 of short duplexes. An inducible knockdown of mRPN1 in T. brucei results in loss of gRNA and accumulation of precursor transcripts (pre-gRNAs), consistent with a role of mRPN1 in processing. mRPN1 stably associates with three proteins previously identified in relatively large complexes that do not contain mRPN1, and have been linked with multiple aspects of mitochondrial RNA metabolism. One protein, TbRGG2, directly binds mRPN1 and is thought to modulate gRNA utilization by editing complexes. The proposed participation of mRPN1 in processing of polycistronic RNA and its specific protein interactions in gRNA expression are discussed.

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Section: Resultssupporting

confidence: 50%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Guide RNA biogenesis involves a novel RNase III family endoribonuclease inTrypanosoma brucei

Madina¹,

Gokulan²,

Vashisht³

et al. 2011

RNA

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“…Plasmids encoding other genes suggested to be involved in trypanosome RNA editing were also included. These are MRP1 and MRP2 (37), mHel61 (38), RBP16 (39), REAP-1 (40), TbRGG1 (16,41), and KRET1 (7,42). The plasmid pools were partially digested with five restriction enzymes that recognize 4-bp sequences, all generating 5Ј-CG overhangs (AciI, HinPII, MaeII, MspI, and TaqI), and fragments ranging up to 2 kb were gel-isolated and ligated into ClaI-digested yeast two-hybrid plasmids pGAD-C1, -C2, and -C3 and pGBD-C1, -C2, and -C3, which are identical except for the translational reading frame of the polylinker region (36).…”

Section: Methodsmentioning

confidence: 99%

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

Schnaufer

Park

et al. 2010

Journal of Biological Chemistry

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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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“…Hybridization was performed in NaP i solution using probes labeled by random priming with [␣-32 P]dATP (MP Biomedicals) overnight at 55°C. Membranes were washed and the radioactive signal was detected as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

The Fe/S Cluster Assembly Protein Isd11 Is Essential for tRNA Thiolation in Trypanosoma brucei

Paris

Changmai

Rubio

et al. 2010

Journal of Biological Chemistry

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Fe/S clusters are part of the active site of many enzymes and are essential for cell viability. In eukaryotes the cysteine desulfurase Nfs (IscS) donates the sulfur during Fe/S cluster assembly and was thought sufficient for this reaction. Moreover, Nfs is indispensable for tRNA thiolation, a modification generally required for tRNA function and protein synthesis. Recently, Isd11 was discovered as an integral part of the Nfs activity at an early step of Fe/S cluster assembly. Here we show, using a combination of genetic, molecular, and biochemical approaches, that Isd11, in line with its strong association with Nfs, is localized in the mitochondrion of T. brucei. In addition to its involvement in Fe/S assembly, Isd11 also partakes in both cytoplasmic and mitochondrial tRNA thiolation, whereas Mtu1, another protein proposed to collaborate with Nfs in tRNA thiolation, is required for this process solely within the mitochondrion. Taken together these data place Isd11 at the center of these sulfur transactions and raises the possibility of a connection between Fe/S metabolism and protein synthesis, helping integrate two seemingly unrelated pathways.

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TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

Cited by 84 publications

References 38 publications

Guide RNA biogenesis involves a novel RNase III family endoribonuclease inTrypanosoma brucei

Guide RNA biogenesis involves a novel RNase III family endoribonuclease inTrypanosoma brucei

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

The Fe/S Cluster Assembly Protein Isd11 Is Essential for tRNA Thiolation in Trypanosoma brucei

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