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

Paris, Zdeněk; Changmai, Piya; Rubio, Mary Anne T.; Zı́ková, Alena; Stuart, Kenneth; Alfonzo, Juan D.; Lukeš, Julius

doi:10.1074/jbc.m109.083774

Cited by 32 publications

(32 citation statements)

References 43 publications

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“…Cell lysates corresponding to 5 × 10 6 cells/lane were separated on 15% SDS-polyacrylamide gel, blotted, and probed. The polyclonal rabbit antibodies against the T. brucei TRM5, Isd11 (Paris et al 2011), Phb1 (Týčet al 2010), MRP1 (Vondrušková et al 2005), and enolase (kindly provided by P.A.M. Michels) were used at 1:100, 1:1.000, 1:1.000, 1:1.000, and 1:1.000 dilutions, respectively. Secondary anti-rabbit IgG antibodies (1:2000) coupled to horseradish peroxidase (GE Healthcare) were visualized according to the manufacturer's protocol using the ECL kit (Pierce).…”

Section: Preparation Of Antibodies and Western Blot Analysismentioning

confidence: 99%

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

Paris

Horáková

Rubio

et al. 2013

RNA

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All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m 1 G 37 ) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m 1 G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m 1 G 37 requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m 1 G 37 , but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species.

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Section: Preparation Of Antibodies and Western Blot Analysismentioning

confidence: 99%

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

Paris

Horáková

Rubio

et al. 2013

RNA

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“…The assembly of a new Fe-S cluster occurs on a large protein complex composed of Nfs, its indispensable interacting partner Isd11, and the scaffold protein Isu (24). In mammals, frataxin is also present in this complex (34), yet this was not confirmed for trypanosomes (27). The newly formed cofactors are transferred by numerous chaperons and scaffold proteins, which either transfer them to the mitochondrial apoproteins or export a so-far-unknown intermediate from the organelle that is further processed and exploited for the cytosolic and nuclear Fe-S-dependent proteins (24)(25)(26).…”

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“…However, no functional data are available on this pathway in the BS other than that the level of its components is much lower than that in the PS, as has been shown for frataxin (35), Isd11 (27), ferredoxin (31), and Isa1 and Isa2 (29). In general, the (in)dispensability of Fe-S machinery in the BS mitochondrion is questionable, since the bulk of Fe-S clusters produced in the organelle of a typical eukaryotic cell are utilized for its respiratory chain complexes, which are either repressed or absent in this life stage of T. brucei.…”

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“…RNA interference (RNAi) depletion of Nfs, Isd11, Isu, Isa1, Isa2, and frataxin resulted in drastic changes in growth phenotypes, decreases in the activity of several Fe-S enzymes, and other downstream effects (27-29, 31, 35). In addition, Nfs and Isd11 are also indispensable for tRNA thiolation (27). Two homologues of the electron donor protein ferredoxin were identified in the genomes of trypanosomatid flagellates, but only ferredoxin A is essential for the Fe-S cluster assembly (31).…”

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“…In addition, Fe-S clusters are utilized in iron metabolism, serve in DNA replication, and have structural or other functions (25). These cofactors are synthesized via a complex pathway composed of more than 20 enzymes, and as detailed below, most of them are present in T. brucei (27)(28)(29)(30)(31).…”

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Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

et al. 2014

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Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

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Transfer RNA modifications: nature's combinatorial chemistry playground

Jackman¹,

Alfonzo²

2012

WIREs RNA

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Following synthesis, tRNAs are peppered by numerous chemical modifications which may differentially affect a tRNA’s structure and function. Although modifications affecting the business ends of a tRNA are predictably important for cell viability, a majority of modifications play more subtle structural roles that can affect tRNA stability and folding. The current trend is that modifications act in concert and it is in the context of the specific sequence of a given tRNA that they impart their differing effects. Recent developments in the modification field have highlighted the diversity of modifications in tRNA. From these the combinatorial nature of modifications in explaining previously described phenotypes derived from their absence has emerged as a growing theme.

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

Cited by 32 publications

References 43 publications

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

Transfer RNA modifications: nature's combinatorial chemistry playground

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