Role of an RNA-binding protein in import of tRNA into Leishmania mitochondria (original) (raw)
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A bifunctional tRNA import receptor from Leishmania mitochondria
2006
In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro. RIC1, the largest subunit of this complex, is structurally homologous to the conserved alpha subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli. Antisense-mediated knockdown of RIC1/F1alpha in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1alpha alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1alpha formed stable complexes with type I, but not type II, tRNAs through the...
An RNA-binding Respiratory Component Mediates Import of Type II tRNAs into Leishmania Mitochondria
Journal of Biological Chemistry, 2006
Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import. Mitochondria from a large number of species, including protists, higher plants, some invertebrates, and eutherian mammals do not contain sufficient numbers of functional tRNA genes and therefore import cytoplasmic tRNAs to support the translation of organellar mRNAs (reviewed in Refs. 1 and 2). Mitochondrial tRNA import is especially important in kinetoplastid protozoa such as Leishmania and Trypanosoma that lack all mitochondrial tRNA genes (3, 4).
Nucleic acids research, 2003
Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA(Tyr) (Type I) transiently stimulates the rate of entry of tRNA(Ile) (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA(Ile). Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA(Tyr) to the D domain, and those of tRNA(Ile) to the variable region-T domain (V-T region), indicating that both activities originate from a single RNA-receptor interaction. A third tRNA, human tRNA(Lys), is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA h...
The D arm of tRNATyr is necessary and sufficient for import into Leishmania mitochondria in vitro
Nucleic Acids Research, 1998
Transfer RNAs are selectively imported from the cytoplasm into mitochondria of kinetoplastid protozoa such as Leishmania. The specific structural features of tRNA which determine selectivity are largely unknown. Using an in organello system from Leishmania, the import signals on tRNA Tyr and on a synthetic transcript which binds to the same receptor, were studied by deletion and reconstruction analyses. In both cases, short oligoribonucleotides (minihelices) containing the sequence UGGYAGAG were imported with high efficiency in the presence of ATP. This motif is present in the D arm of tRNA Tyr , as well as in the majority of imported Leishmania tRNAs. Deletion of the D arm, or a point mutation in the conserved motif, reduces importability. The import signal coincides with the binding site for the mitochondrial receptor TAB. tRNA Gln , which is not imported, forms non-productive, TAB-independent complexes with the mitochondrial surface. However, the observation that the imported:bound ratio of the D arm minihelix is higher than that of the entire molecule suggests that the post-binding translocation step is constrained in terms of size or structural flexibility. Kinetic studies of minihelix import indicate stepwise insertion of the molecule into import channels.
Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex
Nucleic Acids Research, 2008
The RNA import complex (RIC) from the mitochon- drion of the kinetoplastid protozoan Leishmania tropica contains two subunits that directly bind to import signals on two distinct subsets of tRNA and interact with each other allosterically. What happens to the tRNA subsequent to its loading on the complex is unknown. A third subunit—RIC9—has intrinsic affinity for both types of tRNA
Selective importation of RNA into isolated mitochondria from Leishmania tarentolae
RNA, 2000
All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA Ile (UAU) and tRNA Gln (CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA Asp was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than ;17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondrialocalized tRNA Ile as compared to the level of importation of the mainly cytosol-localized tRNA Gln. Furthermore, exchanging the D-arm between the tRNA Ile and the tRNA Gln resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.
RNA, 2002
All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania tarentolae are encoded in the nucleus and are imported from the cytosol into the mitochondrion. We previously reported the partitioning of five tRNAs and found that all were shared between the two compartments to different extents. To increase our knowledge of the tRNAs of this organism, and to attempt to understand the signals involved in their subcellular localization, a method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 39 polyadenylated and reverse transcribed with a sequencetagged primer. The cDNA was tagged by ligation to an anchor oligonucleotide, and the resulting double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing to 20 the total number of L. tarentolae tRNAs identified to date. The subcellular localization of 17 tRNAs was quantitatively analyzed by two-dimensional gel electrophoresis and northern hybridization. In general, the previously suggested operational classification of tRNAs into three groups (mainly cytosolic, mainly mitochondrial, and shared between the two compartments) is still valid, but the relative abundance of each tRNA in the cytosol or mitochondrion varied greatly as did the level of expression.
Embo Journal, 2003
In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA Glu (UUC) and tRNA Gln (UUG) are localized in both cytosol and mitochondria. We investigated structural differences between af®nity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modi®cation difference in both tRNAs was identi®ed at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2thiouridine (mcm 5 s 2 U), whereas mt tRNAs have 5methoxycarbonylmethyl-2¢-O-methyluridine (mcm 5 Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm 5 U) at its wobble position, which could represent a common modi-®cation intermediate for both modi®ed uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-speci®c tRNA Lys (UUU) from the cytosol and found mcm 5 U at its wobble position, while its mitochondrial counterpart has mcm 5 Um. Mt tRNA Lys and in vitro transcribed tRNA Glu were imported much more ef®ciently into isolated mitochondria than the native cy tRNA Glu in an in vitro importation experiment, indicating that cytosol-speci®c 2-thiolation could play an inhibitory role in tRNA import into mitochondria.
Nucleic Acids Research, 1989
The mitochondrion of Leishmania tarentolae contains approximately 35-40 tRNAs, many of which comigrate with cytoplasmic tRNAs. Both mitochondrial (KtRNA) and cytoplasmic (CtRNA) tRNAs are functional, as they could be acylated either by mitochondrial or cytoplasmic synthetase extracts. There are two methionyl tRNA species in the cytoplasmic and mitochondrial fractions, one of which is unique to each fraction, indicating that the KtRNA fraction is free of CtRNA contamination. Leucyl and glycyl tRNAs were identified by hybridization with a genomic clone from Trypanosoma brucei. KtRNA hybridizes with nuclear chromosomes, but not with minicircle or maxicircle DNA. KtRNA isolated by DEAE chromatography or agarose gel electrophoresis contains additional small RNAs which hybridize with both minicircle and maxicircle DNA. These transcripts do not migrate like tRNAs in acrylamide gels and their function is unknown. We suggest that most if not all mitochondrial tRNAs in L. tarentolae are nuclear-encoded and imported into the mitochondrion.