A Single Zinc Ion Is Sufficient for an Active Trypanosoma brucei tRNA Editing Deaminase

Spears, Jessica L.; Rubio, Mary Anne T.; Gaston, Kirk W.; Wywiał, Ewa; Strikoudis, Alexandros; Bujnicki, Janusz M.; Papavasiliou, F. Nina; Alfonzo, Juan D.

doi:10.1074/jbc.m111.243568

Cited by 18 publications

(22 citation statements)

References 33 publications

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“…The resulting expression plasmid encoding 6xHis-tagged TRM5 was transformed into the E. coli strain BL21(DE3) (Novagen). Soluble protein was obtained by IPTG induction of recombinant protein expression in bacterial cells (incubation for 20 h at 25°C) and purified under native conditions using Ni 2+ -chelate chromatography according to the method described previously (Spears et al 2011). …”

Section: Methodsmentioning

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: Methodsmentioning

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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“…This region has canonical zinc binding motifs (HXE, CXXC) (18 -20), which are conserved in deaminases that act on nucleotides, RNA, and DNA (16,(21)(22)(23)(24)(25)(26). The DYW deaminase domain has recently been shown to bind zinc ions (19,20).…”

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confidence: 99%

A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity

Hayes

Dang

Diaz

et al. 2015

Journal of Biological Chemistry

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Background: Pentatricopeptide repeat (PPR) proteins that are required for RNA editing frequently include a C-terminal DYW deaminase domain. Results: Mutagenesis of a glutamate residue in the conserved deaminase HXE motif results in loss of editing activity. Conclusion:The glutamate residue is required for editing. Significance: The DYW deaminase domain of PPR proteins has the molecular characteristics of a deaminase.Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast.RNA editing takes place in most land plant chloroplasts and mitochondria (1, 2). In flowering plants, the transcripts of chloroplasts and mitochondria are modified post-transcriptionally by C-to-U editing with about 35 C-to-U editing events in chloroplasts and hundreds of editing sites in the mitochondria (3).Editing in higher plants and in Physcomitrella patens is known to require nuclear proteins (4 -8).Pentatricopeptide repeat (PPR) 2 genes have been shown to be required for RNA editing (9), and form a large family of protein-coding genes in higher plants with over 400 members in Arabidopsis (10). The known editing factors are members of the PLS subfamily of PPR proteins, which are composed of characteristic P, L (long), and S (short) repeats (11). Amino acid residues located in specific locations within the repeats have been shown to specify the base recognized in the cis-element (12)(13)(14), and the PLS repeat domain interacts with specific nucleotides within the cis-element to provide site specificity for RNA editing (12,15).The PLS subfamily of PPR proteins also includes ...

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“…Although the mechanism for A 34 deamination is similar to that of ADAT1, the two enzymes show marked differences in terms of sub-unit composition as well as at the primary sequence level. The arrangement of active site residues of ADAT2 and ADAT3 resemble those of a cytidine deaminase while residues of ADAT1 resemble those of classical adenosine deaminases [23, 29]. Paradoxically, these enzymes that catalyze A to I editing at position 34 possess all the features of cytosine-specific deaminases.…”

Section: Editing By Deamination: Avoiding Conundrumsmentioning

confidence: 99%

Determinants of tRNA editing and modification: Avoiding conundrums, affecting function

Paris

Fleming

Alfonzo

2012

Seminars in Cell & Developmental Biology

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In all organisms tRNAs play the essential role of connecting the genetic information found in DNA with the protein synthesis machinery ensuring fidelity during translation. Following transcription tRNAs undergo a number of processing events including numerous post-transcriptional modifications that render a tRNA molecule fully functional. The effects of some modifications go beyond simply affecting tRNA structure and can alter the meaning of the tRNA. This review will summarize the current state of the tRNA editing field, highlighting how editing affects tRNA structure and function in various organisms. It will also discuss recent data that hints at connections between editing and modification that may be exploited by cells to modulate a tRNA’s role in translation.

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A Single Zinc Ion Is Sufficient for an Active Trypanosoma brucei tRNA Editing Deaminase

Cited by 18 publications

References 33 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

A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity

Determinants of tRNA editing and modification: Avoiding conundrums, affecting function

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