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• W2157788778 abstract "Article13 September 2007free access Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei Nabile Bouzaidi-Tiali Nabile Bouzaidi-Tiali Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Eric Aeby Eric Aeby Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Fabien Charrière Fabien Charrière Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Mascha Pusnik Mascha Pusnik Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author André Schneider Corresponding Author André Schneider Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Nabile Bouzaidi-Tiali Nabile Bouzaidi-Tiali Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Eric Aeby Eric Aeby Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Fabien Charrière Fabien Charrière Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Mascha Pusnik Mascha Pusnik Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author André Schneider Corresponding Author André Schneider Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland Search for more papers by this author Author Information Nabile Bouzaidi-Tiali1,‡, Eric Aeby1,‡, Fabien Charrière1,‡, Mascha Pusnik1 and André Schneider 1 1Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, Fribourg, Switzerland ‡These authors contributed equally to this work *Corresponding author. Department of Biology, University of Fribourg, Chemin du Musee 10, Fribourg 1700, Switzerland. Tel.: +41 26 300 8877; Fax: +41 26 300 9741; E-mail: [email protected] The EMBO Journal (2007)26:4302-4312https://doi.org/10.1038/sj.emboj.7601857 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mitochondrial tRNA import is widespread in eukaryotes. Yet, the mechanism that determines its specificity is unknown. Previous in vivo experiments using the tRNAsMet, tRNAIle and tRNALys have suggested that the T-stem nucleotide pair 51:63 is the main localization determinant of tRNAs in Trypanosoma brucei. In the cytosol-specific initiator tRNAMet, this nucleotide pair is identical to the main antideterminant that prevents interaction with cytosolic elongation factor (eEF1a). Here we show that ablation of cytosolic eEF1a, but not of initiation factor 2, inhibits mitochondrial import of newly synthesized tRNAs well before translation or growth is affected. tRNASec is the only other cytosol-specific tRNA in T. brucei. It has its own elongation factor and does not bind eEF1a. However, a mutant of the tRNASec expected to bind to eEF1a is imported into mitochondria. This import requires eEF1a and aminoacylation of the tRNA. Thus, for a tRNA to be imported into the mitochondrion of T. brucei, it needs to bind eEF1a, and it is this interaction that mediates the import specificity. Introduction Most protozoa, many fungi, plants and a few animals lack a variable number of mitochondrial tRNA genes. It has been shown in these organisms that the missing genes are compensated for by import of a small fraction of the corresponding cytosolic tRNAs (Schneider and Marechal-Drouard, 2000; Bhattacharyya and Adhya, 2004). The phylogenetic distribution of mitochondrial tRNA import is disperse. Thus, for some species where tRNA import has been predicted, closely related organisms can be found that do not import tRNAs (Schneider and Marechal-Drouard, 2000). Since the loss of mitochondrial tRNA genes is likely to be irreversible, this suggests that the process has a polyphyletic origin. This conclusion is supported by studies of mitochondrial tRNA import in yeast (Tarassov et al, 1995), Leishmania (Goswami et al, 2006) and plants (Salinas et al, 2006), which provided evidence for three distinct tRNA import machineries. The capability to import tRNAs in these three groups of organisms is therefore due to convergent evolution. Consistent with this view is the fact that the number of imported tRNAs is species-specific. Mitochondria of Saccharomyces cerevisiae import two tRNAs only (Tarassov and Martin, 1996; Rinehart et al, 2005). Plants import a variable number of mitochondrial tRNAs, but have retained at least a few mitochondrial tRNA genes (Dietrich et al, 1996b). The most extreme cases are two groups of unrelated parasitic protozoa, the trypanosomatids (which include Trypanosoma brucei and Leishmania spp.) (Simpson et al, 1989; Hancock and Hajduk, 1990; Schneider et al, 1994) and the apicomplexans (Crausaz-Esseiva et al, 2004b), both of which completely lack mitochondrial tRNA genes and therefore must import the whole set of tRNAs. However, in both parasites we still find tRNAs that are cytosol-specific (Crausaz-Esseiva et al, 2004a, 2004b; Geslain et al, 2006). Interestingly, in all organisms that have been analyzed, an imported nucleus-encoded mitochondrial tRNA only represents a small fraction of a normal cytosolic tRNA (Schneider and Marechal-Drouard, 2000; Tan et al, 2002b). Strikingly, the imported fraction is specific for a given tRNA species and varies between 1 and 8%. Thus, two prominent questions regarding mitochondrial targeting of tRNAs are (i) what determines the import specificity and (ii) what regulates the extent of tRNA import? Regarding the latter, it has been suggested that for some leishmanial tRNAs the extent of import is regulated by cytosol-specific thio-modifications in the anticodon (Kaneko et al, 2003). Regarding the former, there are a number of studies in different organisms showing that the import specificity is controlled by localization determinants on mature tRNAs (Rusconi and Cech, 1996; Entelis et al, 1998; Crausaz-Esseiva et al, 2004a). However, as expected due to the polyphyletic origin of tRNA import, they are not identical in the different species. In the imported tRNALys isoacceptor of yeast, the localization signals are confined to the acceptor stem and the anticodon loop, and are required for binding to the precursor of mitochondrial lysyl-tRNA synthetase (Entelis et al, 1998). This protein forms a complex with the imported tRNALys, which then is transported across the mitochondrial membranes by using the protein import pores (Tarassov et al, 1995). It is not known how the other imported yeast tRNA, the tRNAGln, is addressed to mitochondria, and by which mechanism it is imported (Rinehart et al, 2005). The only other species where the in vivo tRNA import determinants have been analyzed in detail are Tetrahymena and T. brucei. For tRNAGln isoacceptors of Tetrahymena it is the anticodon (Rusconi and Cech, 1996), and for the tRNAMet isoacceptors of T. brucei a single T-stem nucleotide pair that are both necessary and sufficient to determine the localization of these tRNAs (Crausaz-Esseiva et al, 2004a). Only fragmentary results are available for what determines the in vivo import specificity in plants; a point mutation in the acceptor stem of tRNAAla of potato was shown to abolish import in vivo (Dietrich et al, 1996a), and more recently the D-loop and the anticodon region were implicated in import of plant tRNAVal (Delage et al, 2003). However, it is not known in any system which factors decode the localization signals. Here we present evidence that in T. brucei, binding to translation elongation factor 1a (eEF1a) is a prerequiste for import, suggesting that it is this interaction that determines the specificity of tRNA import in vivo. Results Correlation between import and binding to EF1a In T. brucei the initiator tRNAMet (Crausaz-Esseiva et al, 2004a) and the tRNASec are cytosol-specific (Geslain et al, 2006). All other tRNAs function in both the cytosol and the mitochondrion (Figure 1). Thus, by expressing chimeras between the closely related cytosolic initiator and the imported elongator tRNAsMet, we showed that the single unmodified T-stem nucleotide pair at position 51:63 is both necessary and sufficient for the correct localization of the tRNAsMet (Crausaz-Esseiva et al, 2004a). The adjacent nucleotide pair 52:62 influences the efficiency of import, but when transplanted onto other tRNAs, was not able to change their localization. Furthermore, we showed that both the cytosolic as well as the mitochondrial localization determinants can act in the context of the tRNAIle and the tRNALys (Crausaz-Esseiva et al, 2004a), suggesting that the same determinants can function in the context of any trypanosomal tRNA. Thus, if we find the T-stem nucleotide pair U51:A63, the tRNA remains in the cytosol, whereas if any other standard base pair, such as C:G, A:U or G:C, is present at this position, the tRNA is in part imported into mitochondria (Crausaz-Esseiva et al, 2004a) (Figure 1). (However, the tRNASec is an exception, despite carrying C51:G63 it is cytosol-specific.) Interestingly, the nucleotide pair U51:A63 is conserved in all eukaryotic initiator tRNAsMet and generally absent from elongator tRNAs. It not only acts as a cytosolic localization determinant in T. brucei, but the corresponding nucleotide pair in vertebrate initiator tRNAMet is one of two antideterminants that prevent binding of cytosolic eEF1a (Drabkin et al, 1998). The trypanosomal eEF1a is 78% identical to its human counterpart (Kaur and Ruben, 1994), which makes it very likely that the U51:A63 nucleotide pair also acts as antideterminant for the T. brucei protein. Furthermore, it has been shown that one tRNA domain recognized by eEF1a is the T-arm (Dreher et al, 1999). Thus, we observe a perfect correlation between mitochondrial import of a given trypanosomal tRNA and its predicted binding to eEF1a. This is not only true for wild-type tRNAs but also for the numerous variants whose localization we have tested in vivo (Crausaz-Esseiva et al, 2004a). In agreement with this correlation we see a congruence of the localization determinant with a nucleotide pair involved in binding or preventing of binding to eEF1a. Based on these observations we suggest the hypothesis that in T. brucei interaction with eEF1a is a prerequisite for a tRNA to be imported into mitochondria, and that it is this binding that determines the specificity of the process. Figure 1.Specificity of mitochondrial tRNA import in T. brucei. Top part: All elongator tRNAs specifying the 20 standard amino acids are in part imported into mitochondria. The signal that determines their localization is the T-stem nucleotide pair C51:63G, A51:U63 or G51:C63. Lower part: The initiator tRNAMet and the tRNASec are cytosol-specific. The cytosolic localization signal of initiator tRNAMet, the nucleotide pair U51:A63 (Crausaz-Esseiva et al, 2004a), is at the same time the major antideterminant for eEF1a binding (Drabkin et al, 1998). The T-stem loop region of the cytosolic tRNASec includes a putative C51:G63 import signal. However, the non-standard U:U nucleotide pair (number 8 in the acceptor stem) is a putative antideterminant for eEF1a binding (Rudinger et al, 1996). Thus, all tRNAs that interact with eEF1a are imported, whereas the ones that do not are cytosol-specific. Download figure Download PowerPoint How does the cytosolic localization of the tRNASec, which lacks the U51:A63 cytosolic localization determinant of the initiator tRNAMet, fit into this picture (Figure 1)? It is known that tRNAsSec do not bind to eEF1a (or the bacterial homologue EF-Tu). In eukaryotes this is most likely due to the non-conventional U:U nucleotide pair at position 9 of the acceptor stem, which acts as an antideterminant for eEF1a binding (Rudinger et al, 1996). tRNAsSec, instead of eEF1a, interact with their own specialized elongation factor, termed EFSec (Diamond, 2004), an orthologue of which has also been identified in T. brucei (Cassago et al, 2006; Lobanov et al, 2006). Taking all this into account, the cytosolic localization of the tRNASec, rather than contradicting our hypothesis, actually supports it. Inducible tRNA expression In order to test the hypothesis that eEF1a is involved in tRNA import, we constructed RNAi cell lines allowing inducible ablation of either eEF1a or as a control of cytosolic translation initiation factor 2 (eIF2). Ablation of both of these proteins, as expected due to their essential function in translation, leads to a growth arrest but did not change the steady-state levels of mitochondrial tRNAs (data not shown). This could however be due to the fact that even in the absence of import the tRNA population that was imported before the induction of RNAi may persist for a long time. It might therefore not be possible to detect an import phenotype by simply analyzing the steady-state population of tRNAs. The very same problem was encountered in the analysis of mitochondrial protein import in yeast, where inducible ablation of a key import factor did not result in an obvious depletion of mitochondrial-localized proteins at steady state (Baker et al, 1990). However, the import phenotype was clearly seen in a pulse–chase experiment, which allows to selectively monitor newly synthesized proteins. Thus, in order to follow the fate of a newly synthesized tRNA in T. brucei, we produced a cell line that allows inducible expression of a nucleus-encoded and imported tRNA. Practically this was achieved by transfection of T. brucei 29-13, which expresses the tetracycline repressor, with a construct containing the tetracycline operator 5′ of a tagged tRNA gene. Figure 2A shows that in these cells addition of tetracycline induces expression of the tagged tRNA in a time-dependent manner. The transgenic tRNA is correctly processed as well as aminoacylated (not shown), and by all means behaves like a fully functional tRNA. Analysis of digitonin-extracted mitochondrial fractions furthermore showed that, as expected, the tagged tRNA was imported into mitochondria. In vitro experiments from different laboratories suggested that tRNA import requires an electrochemical gradient across the mitochondrial inner membrane (Mukherjee et al, 1999; Yermovsky-Kammerer and Hajduk, 1999). The left and the middle panels of Figure 2B show that treatment of a culture of T. brucei with carbonyl cyanide m-chlorophenylhydrazone (CCCP)—an uncoupler that dissipates the electrochemical gradient—inhibits import of the newly synthesized tRNA by 75%. This inhibition is not detected by looking at the steady-state mitochondrial tRNA pool (Figure 2B, left panel), since the major fraction of each tRNA was imported before the CCCP treatment. Staining of cells with Mitotracker (Figure 2B, right panel), a dye that detects the electrochemical gradient, confirms that incubation with CCCP depolarizes the mitochondrial inner membrane and shows that the cells remain alive and morphologically unchanged during the treatment. Figure 2.Tetracycline-inducible expression of a tagged tRNA. (A) Time course of induction. Appearance of the tagged tRNAMet (tRNAMet*) in the cytosol (Tot) and in digitonin-extracted mitochondria (Mit) was monitored by Northern analysis (left side, upper panel). The lower panel shows the corresponding ethidium bromide-stained gel (EtBr). Positions of the mitochondrial rRNAs (Mit rRNA) and the cytosolic rRNAs (Cyt rRNA), as well as the tRNA region are indicated. Graph: Quantitative analysis of four independent experiments of the type shown on the left. The signal corresponding to the tagged tRNAMet at 24 h of induction in the total RNA fraction was set to 100%. Standard errors are indicated. (B) Mitochondrial import of newly synthesized tagged tRNAMet requires the membrane potential. Left panel: Expression of the tagged tRNA was induced for 10 h in absence (−) and presence (+) of 20 mmol of the uncoupler CCCP, and analyzed by Northern blot. Middle panel: Quantitative analysis of four independent experiments of the type shown on the left. The signal in untreated cells that corresponds to the mitochondrially localized tagged tRNAMet after 10 h of induction was set to 100%. Standard errors are indicated. Right panel: Mitotracker-staining of untreated (−) and CCCP-treated cells (+). The y-axis images of the ethidium bromide-stained gels have been electronically compressed by a factor of approximately 2. Download figure Download PowerPoint Thus, these results demonstrate that in vivo import of trypanosomal tRNAs requires an electrochemical gradient across the inner mitochondrial membrane, and provide a proof of principle that inducible tRNA expression can be used to study aspects of mitochondrial tRNA import that previously were not accessible to direct in vivo analysis. Inducible tRNA expression combined with RNAi In a next step we produced two RNAi cell lines, which upon addition of tetracycline, induce the expression of the tagged tRNA gene, and at the same time downregulate the expression of eIF2 or eEF1a, respectively. Both cell lines showed a slow growth phenotype approximately 48 h after induction of RNAi (Figure 3). Furthermore, in both cases, concomitant with the growth arrest a reduction of cytosolic protein synthesis as measured by 35S-methionine incorporation was seen (Figure 3). Figure 3.Inducible tRNA expression combined with RNAi. Growth curve of a representative clonal T. brucei RNAi cell line allowing simultaneously inducible expression of the tagged tRNAMet and ablation of IF2 (ind tRNAMet*/IF2-RNAi) and eEF1a (ind tRNAMet*/EF-RNAi), respectively. Open squares and filled triangles represent growth in the absence or presence of tetracycline, respectively. The inset in the left graph shows a Northern blot for the eIF2 mRNA (IF2). The rRNAs in the lower panel serve as loading controls. The inset in the right panel shows an immunoblot probed for eEF1a (EF), and as control for α-ketoglutarate dehydrogenase (KDH), which is not affected by the RNAi. Quantitation of the signals illustrates that the RNAi causes efficient ablation of eEF1a relative to KDH, reaching 31 and 5% after 24 and 48 h, respectively. The efficiency of cytosolic translation during induction of RNAi expressed by the percentage of 35S-labeled methionine incorporation into total cellular protein is indicated at the bottom of each graph. 35S-labeled methionine incorporation in uninduced cells was set to 100%. Download figure Download PowerPoint However, fractionation of the eEF1a-ablated cell line showed that reproducibly approximately fourfold less of the newly synthesized tRNA was found in the mitochondrial fraction than in uninduced cells (Figure 4A). In contrast, no significant effect on import of the newly synthesized tRNA was detected in cells ablated for eIF2 (Figure 4B). Most importantly, the tRNA import phenotype in the eEF1a cell line is already detected 24 h after induction of RNAi, well before growth or translation is affected (Figure 3). Consistent with this observation, the induced cells are fully motile and the electrochemical gradient of their mitochondria, as evidenced by Mitotracker staining, is identical to the one observed in uninduced cells (data not shown). As a further control, that it is indeed the lack of eEF1a that causes the import phenotype, we tested the import of the newly synthesized tRNA in a cell line ablated for the seryl-tRNA synthetase, an essential protein that as eIF2 and eEF1a is required for translation. It was previously shown that ablation of this enzyme causes deacylation of tRNAsSer and leads to a growth arrest (Supplementary Figure 1A) whose kinetics is identical to the one seen in the eIF2 and eEF1a RNAi cell lines (Geslain et al, 2006) (Figure 3). According to our model we expect that ablation of the seryl-tRNA synthetase, identical to the knockdown of eIF2 but in contrast to the ablation of eEF1a, will not affect the import of the newly synthesized tRNA, which is exactly what is seen (Supplementary Figure 1B). Figure 4.Effect of RNAi on import of newly synthesized tRNA. (A) Presence of the tagged tRNAMet (tRNAMet*) in the cytosol (Tot) and in digitonin-extracted mitochondria (Mit) was monitored by Northern analysis (top panels) in cell lines allowing either inducible expression of the tagged tRNAMet only (ind-tRNAMet*, left panel), or inducible expression of the tagged tRNAMet in combination with ablation of eEF1a (ind tRNAMet*/EF-RNAi, right panel). Relative import efficiency of the newly synthesized tRNAMet are indicated at the bottom. The signal in the total RNA fractions (first line) or the mitochondrial fractions in the control cells (second line) was set to 100%. The lower panels show the corresponding ethidium bromide-stained gels. Positions of the mitochondrial rRNAs (Mit rRNA) and the cytosolic rRNAs (Cyt rRNA), as well as the tRNA region are indicated. (B) Same as panel A, but analysis on the right panel was performed with the cell lines allowing inducible expression of the tagged tRNAMet, in combination with ablation of eIF2 (ind tRNAMet*/IF2-RNAi, right panel). (C) Graph of three independent replicate experiments showing the relative import efficiencies of the newly synthesized tRNA in cells that do not undergo RNAi and in eEF1a and eIF2 ablated cell lines, respectively. Bars=s.e. Download figure Download PowerPoint These results strongly suggest that inhibition of import of newly synthesized tRNAs is a direct consequence of the lack of eEF1a. However, it cannot formally be excluded that the lack of a labile factor, required for tRNA import, that is rapidly degraded under limiting eEF1a concentrations is responsible for inhibition of import. To be imported into mitochondria tRNAs must cross both the nuclear and the mitochondrial membranes. A potential caveat of the in vivo import system is to distinguish nuclear retention from inhibition of mitochondrial import. There are two ways to export tRNAs from the nucleus: the exportin-t and the exportin-5 pathway (Bohnsack et al, 2002; Calado et al, 2002). There is no reason to believe that ablation of eEF1a will affect the exportin-t pathway. At first sight this looks different for the exportin-5 pathway, since it transports both tRNAs and eEF1a. However, nuclear export of eEF1a requires the presence of tRNAs that bind to both eEF1a and exportin-5. Thus, while export of eEF1a depends on tRNAs, the converse is not true and tRNAs are still exported even in the absence of eEF1a (Bohnsack et al, 2002; Calado et al, 2002). Finally, we have addressed this question experimentally for the tRNASec variants that are discussed in the next section. In summary, inhibition of tRNA import by ablation of EF1a shows that in T. brucei eEF1a has a dual function; besides its role in cytosolic translation, it is required for in vivo import of tRNAs into mitochondria and determines the specificity of the process. The tRNASec Eukaryotic and bacterial tRNAsSec do not interact with eEF1a or EF-Tu, respectively (Diamond, 2004). Instead they have their own elongation factors. The cytosolic localization of the trypanosomal tRNASec therefore supports the hypothesis that binding to eEF1a might be a prerequisite for tRNA import. For Escherichia coli tRNASec, the antideterminants for EF-Tu binding have been mapped to the eighth, ninth and tenth base pairs of the acceptor branch (Rudinger et al, 1996). Interestingly, the eighth acceptor stem base pair of eukaryotic tRNASec is invariantly a non-Watson Crick U:U (Figure 5A). It has been suggested, in analogy to the situation in bacteria, that this base pair may act as an antideterminant for eEF1a binding in eukaryotes (Rudinger et al, 1996). Thus, we would expect that a variant of the trypanosomal tRNASec, where the U:U eEF1a antideterminant had been replaced by a standard C:G base pair should bind eEF1a (Figure 5A). Our hypothesis predicts that as a consequence this variant tRNASec should be imported into mitochondria. In transgenic T. brucei cells that express the variant tRNASec, this is indeed observed and sequences derived from the variant tRNASec, contrary to the wild-type tRNASec, are recovered in both the cytosol and the mitochondrial fraction (Figure 5A). However, instead of the intact molecule we reproducibly detect two distinct smaller fragments. Thus, for unknown reasons the tRNASec variant appears to be degraded when present in mitochondria. Figure 5.Mitochondrial import of a tRNASec variants. (A) Predicted secondary structure of the trypanosomal tRNASec. The nucleotide changes that were introduced to obtain the variant tRNASec (var1-tRNASec) that lacks the predicted eEF1a antideterminant are indicated. RNA from the cytosol (Tot) and from digitonin-extracted mitochondria (Mit) of a cell line allowing tetracycline-inducible expression of the variant tRNASec (ind-var1-tRNASec) was analyzed by specific oligonucleotide hybridization for the presence of the wild-type tRNASec (tRNASec) (left panel) and the variant tRNASec (var1-tRNASec) (middle panel). Arrows highlight the two fragments of the variant tRNASec that are reproducibly detected in the mitochondrial fraction. Right panel: Ethidium bromide staining (EtBr) of the corresponding gel. Broken lines indicate which region of the stained gel is represented in the blot. (B) Effect of eEF1a-RNAi on import of newly synthesized var1-tRNASec. Left panel: Northern analysis for var1-tRNASec of cytosolic (Tot) and digitonin-extracted mitochondrial (Mit) RNA fractions of an uninduced (−Tet) and induced (+Tet) cell line that allows tetracycline-regulated expression of the var1-tRNASec in combination with ablation of eEF1a (var1-tRNASec/EF-RNAi). The growth phenotype of this cell line was essentially identical to the one shown for the eEF1a-ablated cell line shown in Figure 3 (data not shown). Bottom panels show a reprobing of the same blot for the endogenous imported tRNAIle. Middle panel: EtBr staining of the corresponding gel. Broken lines indicate which region of the stained gel correspond to which blots. Right panel: Total (Tot), cytosolic (Cyt) and nuclear (Nuc) RNA fractions were analyzed for the presence of var1-tRNASec, the primarily nuclearly localized U6 RNA (U6), the cytosolic initiator tRNAMet (tRNAMet−i) and for tRNAs in general (tRNAs, EtBr). The percentage of the total samples that were analyzed in the different lanes is indicated at the bottom. (C) Predicted secondary structure of the tRNASec. The discriminator nucleotide change that prevents charging by seryl-tRNA synthetase and the nucleotide changes that inactivate the predicted eEF1a antideterminant are indicated. All these changes lead to a variant tRNASec that is termed var2-tRNASec. Left panel: Total RNA from cell lines allowing tetracycline-inducible expression of the var1-tRNASec (ind-var1-tRNASec) and var2-tRNASec (ind-var2-tRNASec), respectively, was analyzed on a long acidic gel. Aminoacylated (aa) and deacylated (da) var1-tRNASec (left lane) and var2-tRNASec (right lane) were detected by specific oligonucleotide hybridization. Middle two panels: RNA from the cytosol (Tot) and from digitonin-extracted mitochondria (Mit) of the var2-tRNASec expressing cell line was analyzed for the presence var2-tRNASec. The corresponding EtBr-stained gel is also shown. Broken lines indicate which region of the stained gel corresponds to which blot. Right panel: Distribution of var2-tRNASec in total, cytosolic and nuclear RNA fractions (as in (B)). Download figure Download PowerPoint In a next experiment we prepared a cell line allowing inducible expression of the tRNASec variant with simultaneous knockdown of eEF1a (Figure 5B). Induction of RNAi led to a similar growth phenotype than is observed in the previously described eEF1a RNAi cell line (Figure 3) (data not shown). As expected according to our model, ablation of eEF1a for 24 h abolished mitochondrial import of the variant tRNASec (Figure 5B, left panel). In order to show that the variant tRNASec accumulates in the cytosol and not in the nucleus, we performed cell fractionations using the detergent digitonin. A quantification of the lanes in the right panel of Figure 5B shows—after normalization to equal cell equivalents—that 50% of the primarily nucleus-localized U6 RNA is recovered in the pellet. However, 88% each of the cytosolic initiator tRNAMet and the variant tRNASec are recovered in the supernatant, confirming that ablation of eEF1a does not interfere with nuclear tRNA export (Figure 5B, right panel). Thus, these experiments directly link import of the variant tRNASec to the presence of eEF1a. Formation of the ternary complex between eEF1a, GTP and tRNA requires the tRNA to be aminoacylated (Ribeiro et al, 1995). We have recently shown that the discriminator nucleotide G73 on tRNASer and the tRNASec is the major identity element recognized by the trypanosomal seryl-tRNA synthetase (Geslain et al, 2006). Thus, changing the G73 on the tRNASec to a C is expected to abolish amin" @default.
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• W2157788778 date "2007-09-13" @default.
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• W2157788778 title "Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei" @default.
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• W2157788778 doi "https://doi.org/10.1038/sj.emboj.7601857" @default.
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