Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
1.
Hum Mutat ; 40(10): 1731-1748, 2019 10.
Article in English | MEDLINE | ID: mdl-31045291

ABSTRACT

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.


Subject(s)
Cardiomyopathy, Hypertrophic/genetics , Genes, Mitochondrial , Genetic Predisposition to Disease , Mutation , Neoplasm Proteins/genetics , RNA Processing, Post-Transcriptional , RNA, Transfer/genetics , Alleles , Amino Acid Substitution , Biomarkers , Cardiomyopathy, Hypertrophic/diagnosis , Cardiomyopathy, Hypertrophic/therapy , Cohort Studies , Enzyme Activation , Female , Gene Expression , Genetic Association Studies , Genotype , Humans , Infant , Kinetics , Male , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Phenotype , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Substrate Specificity
2.
PLoS One ; 12(10): e0186277, 2017.
Article in English | MEDLINE | ID: mdl-29045449

ABSTRACT

The enzyme tRNase Z, a member of the metallo-ß-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.


Subject(s)
Catalysis , Conserved Sequence/genetics , Drosophila Proteins/genetics , Endoribonucleases/genetics , RNA, Transfer/genetics , Alanine/genetics , Amino Acid Substitution/genetics , Animals , Binding Sites , Catalytic Domain , Drosophila melanogaster/enzymology , Saccharomyces cerevisiae/enzymology , Sequence Homology, Amino Acid
3.
PLoS One ; 8(7): e66942, 2013.
Article in English | MEDLINE | ID: mdl-23874404

ABSTRACT

tRNase Z, a member of the metallo-ß-lactamase family, endonucleolytically removes the pre-tRNA 3' trailer in a step central to tRNA maturation. The short form (tRNase Z(S)) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z(L)), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z(L) arose from a tandem duplication of tRNase Z(S) followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z(S) reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z(L) structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z(L) performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z(L) function.


Subject(s)
Endoribonucleases/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Endoribonucleases/chemistry , Humans , Mass Spectrometry , Molecular Sequence Data , Proteolysis , Sequence Homology, Amino Acid
4.
RNA Biol ; 9(3): 283-91, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22336717

ABSTRACT

Numerous mutations in the mitochondrial genome are associated with maternally transmitted diseases and syndromes that affect muscle and other high energy-demand tissues. The mitochondrial genome encodes 13 polypeptides, 2 rRNAs and 22 interspersed tRNAs via long bidirectional polycistronic primary transcripts, requiring precise excision of the tRNAs. Despite making up only ~10% of the mitochondrial genome, tRNA genes harbor most of the pathogenesis-related mutations. tRNase Z endonucleolytically removes the pre-tRNA 3' trailer. The flexible arm of tRNase Z recognizes and binds the elbow (including the T-loop) of pre-tRNA. Pathogenesis-related T-loop mutations in mitochondrial tRNAs could thus affect tRNA structure, reduce tRNase Z binding and 3' processing, and consequently slow mitochondrial protein synthesis. Here we inspect the effects of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs on pre-tRNA structure and tRNase Z processing. Increases in K(M) arising from 59A > G substitutions in mitochondrial tRNA(Gly) and tRNA(Ile) accompany changes in T-loop structure, suggesting impaired substrate binding to enzyme.


Subject(s)
Mutation , Nucleic Acid Conformation , RNA 3' End Processing , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA/genetics , RNA/metabolism , Base Sequence , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Humans , Models, Molecular , Molecular Sequence Data , RNA/chemistry , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Mitochondrial , RNA, Transfer/chemistry
5.
J Biol Chem ; 284(23): 15685-91, 2009 Jun 05.
Article in English | MEDLINE | ID: mdl-19351879

ABSTRACT

tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3' end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured alphaalphabetabeta hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (alpha4-alpha5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the beta10-beta11 loop.


Subject(s)
Drosophila Proteins/genetics , Endoribonucleases/genetics , Amino Acid Sequence , Animals , Bacteria/enzymology , Bacteria/genetics , Conserved Sequence , DNA Primers , Drosophila/enzymology , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Genetic Variation , Glycine , Humans , Kinetics , Methionine , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment
6.
RNA Biol ; 5(2): 104-11, 2008.
Article in English | MEDLINE | ID: mdl-18421255

ABSTRACT

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. In the course of tRNA maturation, RNase P removes the 5' end leader and tRNase Z can endonucleolytically remove the 3' end trailer. A domain remote from the active site of tRNase Z recognizes and binds substrate, principally through contacts with the elbow (D/T loops) of the tRNA. To evaluate possible contacts, processing kinetics was performed using human nuclear encoded pre-tRNA(Arg) with substitutions in conserved D and T loop nucleotides. Changes in K(M) observed with some of the substitutions suggest contacts between tRNase Z and substrate tRNA in this region, and changes in tRNA structure provide an additional basis for interpretation of the kinetic effects.


Subject(s)
Conserved Sequence , Endoribonucleases/metabolism , Mutation/genetics , Nucleic Acid Conformation , RNA Precursors/chemistry , RNA Precursors/genetics , Bacillus subtilis/enzymology , Base Sequence , Catalysis , Humans , Kinetics , Molecular Sequence Data , RNA Processing, Post-Transcriptional , RNA, Transfer, Arg/chemistry , RNA, Transfer, Arg/genetics , RNA, Transfer, Val/chemistry , RNA, Transfer, Val/genetics , Substrate Specificity
7.
Biochemistry ; 46(33): 9380-7, 2007 Aug 21.
Article in English | MEDLINE | ID: mdl-17655328

ABSTRACT

tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.


Subject(s)
Endoribonucleases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Catalysis , Conserved Sequence , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Endoribonucleases/genetics , Histidine/chemistry , Histidine/genetics , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Alignment
8.
RNA ; 12(6): 1104-15, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16618969

ABSTRACT

tRNase Z, which can endonucleolytically remove pre-tRNA 3'-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the beta-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k(cat) without affecting k(M), five- to 15-fold increases in k(M) are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k(M) suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3' end approximately 80 times less efficiently than a pre-tRNA possessing natural sequence of the 3'-end trailer, due to reduced k(cat) with no effect on k(M), showing the CCA anti-determinant to be a characteristic of this enzyme.


Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Transfer/metabolism , Alanine/genetics , Alanine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Arginine/genetics , Arginine/metabolism , Bacillus subtilis/enzymology , Drosophila Proteins/genetics , Endoribonucleases/genetics , Glycine/genetics , Glycine/metabolism , Histidine/chemistry , Histidine/genetics , Molecular Sequence Data , Point Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Precursors/genetics , Sequence Alignment , Sequence Homology, Amino Acid , beta-Lactamases/metabolism
9.
J Biol Chem ; 281(7): 3926-35, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16361254

ABSTRACT

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. The 5' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3' end trailer. Long ((L)) and short ((S)) forms of the tRNase Z gene are present in the human genome. tRNase Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA(Ser(UCN)) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.


Subject(s)
Endoribonucleases/metabolism , Mitochondria/metabolism , Mutation , RNA Precursors/genetics , RNA, Transfer, Ser/genetics , RNA/genetics , Humans , Kinetics , Mitochondrial Diseases/etiology , RNA Precursors/chemistry , RNA, Mitochondrial , Substrate Specificity
10.
J Mol Biol ; 350(2): 189-99, 2005 Jul 08.
Article in English | MEDLINE | ID: mdl-15935379

ABSTRACT

Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the beta-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (approximately 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (approximately 500-5000-fold). Wild-type and mutant dissociation constants (Kd values), determined using gel shifts, displayed no substantial differences, and were of the same order as kM (2-20 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced kcat values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein.


Subject(s)
Conserved Sequence , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Histidine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Electrophoretic Mobility Shift Assay , Endoribonucleases/genetics , Kinetics , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , Substrate Specificity
11.
Nucleic Acids Res ; 32(18): 5430-41, 2004.
Article in English | MEDLINE | ID: mdl-15477393

ABSTRACT

Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3' end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3' end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3' end metabolism (3' end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases.


Subject(s)
3' Untranslated Regions/metabolism , Mitochondrial Diseases/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA/genetics , RNA/metabolism , 3' Untranslated Regions/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Base Sequence , Endoribonucleases/metabolism , Humans , Mitochondrial Diseases/metabolism , Molecular Sequence Data , Point Mutation , RNA/chemistry , RNA 3' End Processing , RNA Nucleotidyltransferases/metabolism , RNA, Mitochondrial , RNA, Transfer/chemistry
12.
Biochem Biophys Res Commun ; 322(3): 803-13, 2004 Sep 24.
Article in English | MEDLINE | ID: mdl-15336535

ABSTRACT

The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA(Ser(UCN)) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA(Ser(UCN)) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3' processing endonuclease (tRNase Z) confirmed that the efficiency of both 5' and 3' processing was impaired. A mutant precursor not already processed at the 5' end was poorly processed in vitro by tRNase Z. Misprocessing at the 3' end further impaired the efficiency and accuracy of 5' processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.


Subject(s)
DNA, Mitochondrial/genetics , RNA, Transfer, Ser/genetics , 5' Untranslated Regions/genetics , Base Sequence , Bone Neoplasms , Cell Line, Tumor , DNA Primers , Humans , Molecular Sequence Data , Mutagenesis , Nucleic Acid Conformation , Osteosarcoma , RNA Processing, Post-Transcriptional/genetics , Reverse Transcriptase Polymerase Chain Reaction
13.
J Mol Biol ; 337(3): 535-44, 2004 Mar 26.
Article in English | MEDLINE | ID: mdl-15019775

ABSTRACT

Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.


Subject(s)
Mitochondrial Diseases/genetics , Point Mutation , RNA 3' End Processing , RNA, Transfer, Leu/genetics , RNA/genetics , Humans , Kinetics , Nucleic Acid Conformation , RNA/physiology , RNA Nucleotidyltransferases/metabolism , RNA, Mitochondrial , RNA, Transfer, Leu/physiology
14.
Nucleic Acids Res ; 32(1): 255-62, 2004.
Article in English | MEDLINE | ID: mdl-14715923

ABSTRACT

Although correct tRNA 3' ends are crucial for protein biosynthesis, generation of mature tRNA 3' ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3' end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3' endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3' end processing. In addition our data show that in vivo 5' processing of nuclear and mitochondrial pre-tRNAs occurs before 3' processing.


Subject(s)
Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Endoribonucleases/metabolism , RNA 3' End Processing , RNA Precursors/metabolism , RNA, Transfer/metabolism , RNA/metabolism , Amino Acid Sequence , Animals , Catalysis , Cell Nucleus/genetics , Down-Regulation , Drosophila melanogaster/cytology , Endoribonucleases/chemistry , Endoribonucleases/genetics , Molecular Sequence Data , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA Interference , RNA Precursors/chemistry , RNA Precursors/genetics , RNA, Mitochondrial , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribonuclease P/metabolism , Substrate Specificity
15.
Nucleic Acids Res ; 31(7): 1904-12, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12655007

ABSTRACT

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA(Ile) precursor 3' end maturation in vitro by 3'-tRNase. Strikingly, four pathogenic tRNA(Ile) mutations reduce 3'-tRNase processing efficiency (V(max) / K(M)) to approximately 10-fold below that of wild-type, principally due to lower V(max). The structural impact of mutations was sought by secondary structure probing and wild-type tRNA(Ile) precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA(Ile) precursors with the greatest 3' end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA(Ile) precursor 3' end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.


Subject(s)
DNA, Mitochondrial/genetics , RNA Processing, Post-Transcriptional , RNA, Transfer, Ile/genetics , Base Sequence , Endoribonucleases/metabolism , HeLa Cells , Humans , Kinetics , Molecular Sequence Data , Mutation , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Transfer, Ile/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...