Final Report Summary - CHLAMITRNA (Dissecting the cytosolic tRNA import process in mitochondria of the unicellular green alga Chlamydomonas reinhardtii)
Mitochondria are organelles found in almost all eukaryotic cells. They contain a genetic system with a genome that encodes a number of proteins-coding genes involved in molecular respiration, which generate most of the cell's energy. The synthesis of these mitochondrial-encoded proteins is thus essential for life and requires a complete set of transfer RNAs (tRNAs). In several organisms, the number of mitochondrial tRNA genes is not sufficient to ensure mitochondrial translation. Consequently, nuclear encoded tRNAs have to be imported from the cytosol to mitochondria. Initially, tRNA import in mitochondria was thought to be restricted to only a few taxa of eukaryotes.
Today, it has been recognised that mitochondrial tRNA import is a widespread phenomenon. It has been experimentally documented in diverse organisms including protozoa, fungi, higher plants and most recently in humans. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary. The research made during this intra-European fellowship represents the first comprehensive study on tRNA import in the model green algae Chlamydomonas reinhardtii.
This study was divided into two parts: the first aspect concerns the regulation of this process and the second aspect concerns the tRNA import machinery.
1. Study of the mitochondrial tRNA import regulation
The number of cytosolic tRNAs imported into mitochondria varies from only one in marsupials to the full set as seen in T. brucei. However, in all species a number of tRNAs are exclusively cytosolic indicating the need of the cell to discriminate between imported tRNAs and cytosolic tRNAs. A particular feature in plants is that the number and the identity of imported tRNAs also vary from one specie to another, even in closely related species. Since the tRNA sequences are highly conserved in plants, it remains difficult to understand how plants can differentially regulate the import of identical tRNAs. Interestingly, what was observed is that the mitochondrial population of nuclear-encoded tRNAs is primarily complementary to those encoded in the mitochondrial genome.
Chlamydomonas reinhardtii is a fresh water biflagellated unicellular green alga. Its linear mitochondrial genome only encodes three tRNAs. As a consequence, the mitochondrial translation machinery depends on the import of nucleus-encoded tRNAs. Out of the 49, 34 cytosolic tRNA issoacceptors are present within mitochondria. In Chlamydomonas this process is highly specific. Only essential cytosolic tRNAs are present in mitochondria as observed in higher plants. But in this organism an additional layer of regulation exists. Indeed, the steady-state levels of nuclear-encoded tRNAs in cytosol are correlated with the frequency of occurrence of the corresponding codons in nuclear genes. Fascinatingly and this is the first time observed in any organism, in Chlamydomonas, the levels of imported tRNAs appear also to correlate with the frequency of codons used in mitochondrial genes. As the nuclear and the mitochondrial codon usages are biased, a differential mitochondrial localisation of tRNAs is required in order to allow optimal mitochondrial translation. As a matter of fact, the extent of mitochondrial localisation for each tRNA species was shown to be highly variable, ranging from 0.2 % to 98 %.
All these data taken together suggest that, the identity and the levels of imported tRNAs, can be somehow defined by the information contained in the mitochondrial genome. In order to test this hypothesis our strategy has been to modify the Chlamydomonas mitochondrial genome and observe the repercussions on tRNA import.
1.1 Modification of the mitochondrial tRNA gene content
A first approach to address this question was to insert in the mitochondrial genome of Chlamydomonas, a tagged tRNAVal(UAC) gene corresponding to an efficiently imported tRNA. Genetic transformation of Chlamydomonas mitochondria was achieved by biolistic methods. A vector with the mitochondrial DNA region containing cox1 wild-type gene and the tagged tRNAVal(UAC) was constructed. This construct was then used for mitochondrial transformation of a Chlamydomonas cox1 mutant (dum18) that has lost the capacity to grow in the dark. After transformation, Chlamydomonas recipient strains were placed in the dark, to select transformants that potentially recovered the modified DNA. After 2 months in the dark, 300 transformants were recovered and have been analysed for the presence of the tagged tRNAVal(UAC) gene. The screening revealed only one transformant with the tagged tRNA and the expression of the inserted tRNA gene was demonstrated. Unfortunately, genetic data and Southern blot experiments on mitochondrial DNA of this mutant showed that the insertion was nuclear and not mitochondrial. However, even if the initial objective was not reached, it is very interesting to observe that the tagged tRNA can be expressed in the nuclear context. We are currently investigating whether this additional exogenous tRNA is imported in mitochondria or not and if its expression or import affects the import of the wild-type tRNAVal(UAC).
1.2 Modification of mitochondrial codon usages in nd4 and nd5 mitochondrial genes
A second approach to address this question was to determine whether the replacement in mitochondrial genes of an often-used codon by a seldom-used codon could influence the import efficiency of the corresponding cytosolic tRNA species. For that purpose, both tRNAGly(GCC) and tRNAGly(CCC) were used as models: The tRNAGly(GCC) is efficiently imported into mitochondria (3.6 %). This tRNA enables the decoding of both (GGC) and (GGT) codons that represent 7.5 % of all the codons in mitochondria. In contrast, tRNAGly(CCC) is poorly imported into mitochondria (0.15 %) and the corresponding codon (GGG) only represents 0.08 % of total codons. Thus, we modified the cloned version of the mitochondrial nd4 and nd5 genes by replacing (GGC/GGT) codons by (GGG) codons. The modified versions of the nd4 and nd5 genes were introduced in Chlamydomonas cells by mitochondrial transformation of a strain deleted for the cob gene (dum11) and of a strain deleted of cob and the 3' end of nd4 gene (dum22). After two months in the dark, 500 transformants were recovered with the dum11 strain and only one transformant was recovered with the dum22 strain. The screening of transformed dum11 strain revealed two transformants with only modifications in the nd4 gene. The sequencing of the nd4 gene of these 2 transformants showed 6 and 9 changes respectively instead of 25 expected modifications. This sequencing also showed that these two transformants were at the heteroplasmic state and they could not been purified to homogeneity by subcloning which is without precedent. The sequencing of the transformed dum22 revealed 11 changes in the nd4 gene. Thus, to sum up, we obtained 3 transformants in which the codon usage of (GGG) is varying from 0.08 % in wild type to 0.14 %, 0.17 % and 0.19 %. The import status of tRNAGly(CCC) is currently investigated.
Beyond tRNA import, it is important to point out that it is the first time that a stable heteroplasmic mitochondrial mutant could be obtained. Furthermore, the fact that we have obtained mutants with only few modifications must have a meaning. The precise biological significance of these findings remains to be elucidated, although it is imaginable that the number of modifications cannot exceed a particular threshold without being lethal. The tools now in our hands will with no doubt be invaluable for future studies.
2. Study of VDAC proteins in Chlamydomonas reinhardtii
In higher plants, the voltage dependent anion channel (VDAC) is the major component of the tRNA transport through the outer mitochondrial membrane. Since Chlamydomonas belongs to the green lineage, the homologous VDAC proteins are likely to be involved in tRNA import processes as in higher plants. In this organism, two genes encode VDAC-like proteins namely VDACI and VDACII. Therefore, it was interesting to explore in vivo the role of each VDAC proteins in tRNA import, to see whether these two proteins have or not a differential function in mitochondria and to better understand the role of these two VDAC proteins in tRNA translocation. To do so, RNA silencing was used to downregulate VDAC proteins. We have obtained transformants showing reduced expression of VDACI and VDACII. We analysed the physiological activities i.e. respiration and enzyme activity analysis of mitochondrial respiratory complexes and the activity of complex I appears to be affected in these mutants (35 % less activity compared to the wild type). The work continues so as to obtain a mutant affected in both VDACI and VDACII. The tRNA content of mitochondria of the 3 mutants will be analysed in order to see if tRNA import has been affected.%
L3. Potential impact and use
The research made during this intra-European fellowship comes as a first comprehensive study on tRNA import in Chlamydomonas reinhardtii. The understanding of this mechanism, and in general, mitochondrial nucleic acids import has important technological applications. Indeed, the knowledge obtained on the comprehension of this process in Chlamydomonas is not only a fundamental cellular process of macromolecular traffic between cellular compartments but it also holds promise for development of strategies to address nucleic acids into mitochondria of organisms in which mitochondrial transformation is still difficult or even impossible as in the case of higher plants. For instance, this knowledge could offer the development of strategies for therapeutic interventions in human genetic diseases caused by mutations in mitochondrial tRNA genes.
Today, it has been recognised that mitochondrial tRNA import is a widespread phenomenon. It has been experimentally documented in diverse organisms including protozoa, fungi, higher plants and most recently in humans. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary. The research made during this intra-European fellowship represents the first comprehensive study on tRNA import in the model green algae Chlamydomonas reinhardtii.
This study was divided into two parts: the first aspect concerns the regulation of this process and the second aspect concerns the tRNA import machinery.
1. Study of the mitochondrial tRNA import regulation
The number of cytosolic tRNAs imported into mitochondria varies from only one in marsupials to the full set as seen in T. brucei. However, in all species a number of tRNAs are exclusively cytosolic indicating the need of the cell to discriminate between imported tRNAs and cytosolic tRNAs. A particular feature in plants is that the number and the identity of imported tRNAs also vary from one specie to another, even in closely related species. Since the tRNA sequences are highly conserved in plants, it remains difficult to understand how plants can differentially regulate the import of identical tRNAs. Interestingly, what was observed is that the mitochondrial population of nuclear-encoded tRNAs is primarily complementary to those encoded in the mitochondrial genome.
Chlamydomonas reinhardtii is a fresh water biflagellated unicellular green alga. Its linear mitochondrial genome only encodes three tRNAs. As a consequence, the mitochondrial translation machinery depends on the import of nucleus-encoded tRNAs. Out of the 49, 34 cytosolic tRNA issoacceptors are present within mitochondria. In Chlamydomonas this process is highly specific. Only essential cytosolic tRNAs are present in mitochondria as observed in higher plants. But in this organism an additional layer of regulation exists. Indeed, the steady-state levels of nuclear-encoded tRNAs in cytosol are correlated with the frequency of occurrence of the corresponding codons in nuclear genes. Fascinatingly and this is the first time observed in any organism, in Chlamydomonas, the levels of imported tRNAs appear also to correlate with the frequency of codons used in mitochondrial genes. As the nuclear and the mitochondrial codon usages are biased, a differential mitochondrial localisation of tRNAs is required in order to allow optimal mitochondrial translation. As a matter of fact, the extent of mitochondrial localisation for each tRNA species was shown to be highly variable, ranging from 0.2 % to 98 %.
All these data taken together suggest that, the identity and the levels of imported tRNAs, can be somehow defined by the information contained in the mitochondrial genome. In order to test this hypothesis our strategy has been to modify the Chlamydomonas mitochondrial genome and observe the repercussions on tRNA import.
1.1 Modification of the mitochondrial tRNA gene content
A first approach to address this question was to insert in the mitochondrial genome of Chlamydomonas, a tagged tRNAVal(UAC) gene corresponding to an efficiently imported tRNA. Genetic transformation of Chlamydomonas mitochondria was achieved by biolistic methods. A vector with the mitochondrial DNA region containing cox1 wild-type gene and the tagged tRNAVal(UAC) was constructed. This construct was then used for mitochondrial transformation of a Chlamydomonas cox1 mutant (dum18) that has lost the capacity to grow in the dark. After transformation, Chlamydomonas recipient strains were placed in the dark, to select transformants that potentially recovered the modified DNA. After 2 months in the dark, 300 transformants were recovered and have been analysed for the presence of the tagged tRNAVal(UAC) gene. The screening revealed only one transformant with the tagged tRNA and the expression of the inserted tRNA gene was demonstrated. Unfortunately, genetic data and Southern blot experiments on mitochondrial DNA of this mutant showed that the insertion was nuclear and not mitochondrial. However, even if the initial objective was not reached, it is very interesting to observe that the tagged tRNA can be expressed in the nuclear context. We are currently investigating whether this additional exogenous tRNA is imported in mitochondria or not and if its expression or import affects the import of the wild-type tRNAVal(UAC).
1.2 Modification of mitochondrial codon usages in nd4 and nd5 mitochondrial genes
A second approach to address this question was to determine whether the replacement in mitochondrial genes of an often-used codon by a seldom-used codon could influence the import efficiency of the corresponding cytosolic tRNA species. For that purpose, both tRNAGly(GCC) and tRNAGly(CCC) were used as models: The tRNAGly(GCC) is efficiently imported into mitochondria (3.6 %). This tRNA enables the decoding of both (GGC) and (GGT) codons that represent 7.5 % of all the codons in mitochondria. In contrast, tRNAGly(CCC) is poorly imported into mitochondria (0.15 %) and the corresponding codon (GGG) only represents 0.08 % of total codons. Thus, we modified the cloned version of the mitochondrial nd4 and nd5 genes by replacing (GGC/GGT) codons by (GGG) codons. The modified versions of the nd4 and nd5 genes were introduced in Chlamydomonas cells by mitochondrial transformation of a strain deleted for the cob gene (dum11) and of a strain deleted of cob and the 3' end of nd4 gene (dum22). After two months in the dark, 500 transformants were recovered with the dum11 strain and only one transformant was recovered with the dum22 strain. The screening of transformed dum11 strain revealed two transformants with only modifications in the nd4 gene. The sequencing of the nd4 gene of these 2 transformants showed 6 and 9 changes respectively instead of 25 expected modifications. This sequencing also showed that these two transformants were at the heteroplasmic state and they could not been purified to homogeneity by subcloning which is without precedent. The sequencing of the transformed dum22 revealed 11 changes in the nd4 gene. Thus, to sum up, we obtained 3 transformants in which the codon usage of (GGG) is varying from 0.08 % in wild type to 0.14 %, 0.17 % and 0.19 %. The import status of tRNAGly(CCC) is currently investigated.
Beyond tRNA import, it is important to point out that it is the first time that a stable heteroplasmic mitochondrial mutant could be obtained. Furthermore, the fact that we have obtained mutants with only few modifications must have a meaning. The precise biological significance of these findings remains to be elucidated, although it is imaginable that the number of modifications cannot exceed a particular threshold without being lethal. The tools now in our hands will with no doubt be invaluable for future studies.
2. Study of VDAC proteins in Chlamydomonas reinhardtii
In higher plants, the voltage dependent anion channel (VDAC) is the major component of the tRNA transport through the outer mitochondrial membrane. Since Chlamydomonas belongs to the green lineage, the homologous VDAC proteins are likely to be involved in tRNA import processes as in higher plants. In this organism, two genes encode VDAC-like proteins namely VDACI and VDACII. Therefore, it was interesting to explore in vivo the role of each VDAC proteins in tRNA import, to see whether these two proteins have or not a differential function in mitochondria and to better understand the role of these two VDAC proteins in tRNA translocation. To do so, RNA silencing was used to downregulate VDAC proteins. We have obtained transformants showing reduced expression of VDACI and VDACII. We analysed the physiological activities i.e. respiration and enzyme activity analysis of mitochondrial respiratory complexes and the activity of complex I appears to be affected in these mutants (35 % less activity compared to the wild type). The work continues so as to obtain a mutant affected in both VDACI and VDACII. The tRNA content of mitochondria of the 3 mutants will be analysed in order to see if tRNA import has been affected.%
L3. Potential impact and use
The research made during this intra-European fellowship comes as a first comprehensive study on tRNA import in Chlamydomonas reinhardtii. The understanding of this mechanism, and in general, mitochondrial nucleic acids import has important technological applications. Indeed, the knowledge obtained on the comprehension of this process in Chlamydomonas is not only a fundamental cellular process of macromolecular traffic between cellular compartments but it also holds promise for development of strategies to address nucleic acids into mitochondria of organisms in which mitochondrial transformation is still difficult or even impossible as in the case of higher plants. For instance, this knowledge could offer the development of strategies for therapeutic interventions in human genetic diseases caused by mutations in mitochondrial tRNA genes.
