Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 86
Filter
1.
Int J Biochem Cell Biol ; 34(11): 1502-5, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12200043

ABSTRACT

Molecular misreading is an expression used to describe errors in RNA that lead to the translation of mutated proteins. We have shown that dinucleotide deletions (delta GA, delta GU) are introduced in simple sequence repeats (e.g. GAGAG) of mRNA. If the resulting mutant transcripts escape RNA quality control systems, they are translated into +1 proteins. If functional domains are located downstream of the frameshift site, the result will be a protein with either a partial or complete loss of function. A clear example is ubiquitin(+1) (UBB(+1)), which has lost its capacity to ubiquitinate, i.e. tagging proteins destined for proteasomal degradation. This is an important step in regulating the degradation of misfolded proteins and transcription factors. In fact, UBB(+1) seems to block the proteasome. UBB(+1) and other proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD), which suggests a causal relationship. We have hypothesized that quality control mechanisms for both transcripts and proteins work less efficiently during aging. In this manner +1 proteins may become manifest and contribute to age-related diseases.


Subject(s)
Aging/physiology , Frameshift Mutation , Ubiquitin/genetics , Alzheimer Disease/physiopathology , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ubiquitin/metabolism
2.
Nucleic Acids Res ; 29(14): 2950-62, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11452020

ABSTRACT

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.


Subject(s)
Crithidia fasciculata/genetics , Protozoan Proteins , RNA-Binding Proteins/genetics , RNA/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Molecular Sequence Data , Precipitin Tests , Protein Binding , RNA, Guide, Kinetoplastida/metabolism , RNA, Messenger/metabolism , RNA, Mitochondrial , RNA, Protozoan/metabolism , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Trypanosoma brucei brucei/genetics
3.
Neurobiol Aging ; 21(6): 879-91, 2000.
Article in English | MEDLINE | ID: mdl-11124436

ABSTRACT

Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called "+1 proteins" are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an aberrant C-terminus downstream from the site of the dinucleotide deletion. Molecular misreading was discovered in the rat vasopressin gene associated with diabetes insipidus and subsequently in human genes linked to Alzheimer's disease (AD), e.g. beta amyloid precursor protein (betaAPP) and ubiquitin-B (UBB). Furthermore, betaAPP(+1) and UBB(+1) proteins accumulate in the neuropathological hallmarks (i.e. in the tangles, neuritic plaques, and neuropil threads) of AD. As these +1 proteins were also found in elderly nondemented controls, but not in younger ones (<51 years), molecular misreading in nondividing cells might act as a factor that only becomes manifest at an advanced age. Frameshift mutations (UBB(+1)) and pretangle staining (Alz-50 and MC1) seem to occur independently of each other during early stages of AD. We recently detected +1 proteins, not only in proliferating cells present in non-neuronal tissues such as the liver and epididymis, but also in neuroblastoma cell lines. These observations suggest that molecular misreading is a general source of transcript errors that are involved in cellular derangements in various age-related pathologies.


Subject(s)
Aging/genetics , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Frameshift Mutation , Gene Expression Regulation, Developmental , Sequence Deletion , Transcription, Genetic , Animals , Diabetes Insipidus/genetics , Humans , RNA, Messenger/genetics , Rats
4.
Ann N Y Acad Sci ; 908: 267-81, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10911966

ABSTRACT

Molecular misreading is a novel process that causes mutations in neuronal transcripts. It is defined as the inaccurate conversion of genomic information from DNA into nonsense transcripts and the subsequent translation into mutant proteins. As a result of dinucleotide deletions (delta GA, delta GU, delta CU) in and around GAGAG motifs in mRNA the reading frame shifts to the +1 frame, and subsequently the so-called +1 proteins are synthetized. +1 Proteins have a wild-type NH2 terminus and from the site of the dinucleotide deletion onwards an aberrant, nonfunctional COOH terminus. Molecular misreading was found in the rat vasopressin gene associated with diabetes insipidus and in the human genes linked to Alzheimer's disease (AD), that is, beta-amyloid precursor protein (beta APP) and ubiquitin-B (UBB). Moreover, beta APP+1 and UBB+1 proteins accumulate in the neuropathological hallmarks of AD. Inasmuch as these +1 proteins were also found in elderly, nondemented control patients, but not in younger ones (< 72 years), molecular misreading may act as a factor that becomes manifest in aged people. A hotspot for dinucleotide deletions is GAGAG motifs. Because statistically an average of 2.1 GAGAG motifs per gene can be expected, other genes expressed in other tissues may undergo molecular misreading as well. Indeed, we recently detected +1 proteins in proliferating cells present in tissues such as the liver, epididymis, parotid gland, and neuroblastoma cell lines. Therefore, molecular misreading can be regarded as a general biological source of transcript errors that may be involved in cellular derangements in numerous age-related pathologic conditions apart from Alzheimer's disease.


Subject(s)
Aging/genetics , Mutation , Transcription, Genetic , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Dementia/genetics , Down Syndrome/genetics , Humans , Neurodegenerative Diseases/genetics , Open Reading Frames , RNA/metabolism , Rats , Ubiquitins/genetics
5.
RNA ; 6(1): 121-35, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10668805

ABSTRACT

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.


Subject(s)
DNA, Kinetoplast/genetics , RNA, Guide, Kinetoplastida/genetics , Trypanosomatina/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Kinetoplast/ultrastructure , Evolution, Molecular , Microscopy, Electron , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Trypanosomatina/metabolism , Trypanosomatina/ultrastructure
6.
Nucleic Acids Res ; 28(1): 148-52, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10592207

ABSTRACT

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbas e.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.


Subject(s)
DNA, Mitochondrial/genetics , Databases, Factual , Animals , DNA, Fungal/genetics , DNA, Plant/genetics , Eukaryota/genetics , Humans , Internet , Invertebrates/genetics , Plants/genetics , Vertebrates/genetics
7.
Nucleic Acids Res ; 27(1): 128-33, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-9847157

ABSTRACT

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute. The database can be accessed on the Web at the following address: http://www.ebi.ac. uk/htbin/Mitbase/mitbase.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecie diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.


Subject(s)
DNA, Mitochondrial/genetics , Databases, Factual , Animals , Cell Nucleus/genetics , Classification , DNA, Mitochondrial/classification , Eukaryota/genetics , Europe , Fungi/genetics , Genetic Code , Genetic Diseases, Inborn/genetics , Genetic Variation , Humans , Information Storage and Retrieval , Internet , Invertebrates/genetics , Mutation , Plants/genetics , User-Computer Interface , Vertebrates/genetics
8.
J Biol Chem ; 273(34): 21825-33, 1998 Aug 21.
Article in English | MEDLINE | ID: mdl-9705321

ABSTRACT

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).


Subject(s)
Mitochondria/chemistry , Poly U/metabolism , Protozoan Proteins , RNA Editing , RNA, Protozoan/biosynthesis , RNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/chemistry , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Molecular Weight , Protein Binding , RNA/metabolism , RNA, Mitochondrial , RNA, Ribosomal/biosynthesis , RNA-Binding Proteins/genetics
9.
Nucleic Acids Res ; 26(5): 1205-13, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9469817

ABSTRACT

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle.


Subject(s)
Kinetoplastida/genetics , Kinetoplastida/metabolism , RNA Editing , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA, Kinetoplast/genetics , DNA, Mitochondrial/genetics , Electron Transport Complex IV/genetics , Evolution, Molecular , Kinetoplastida/ultrastructure , Microscopy, Electron , Molecular Sequence Data , NADH Dehydrogenase/genetics , Phylogeny , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Trypanosoma brucei brucei/genetics
10.
Biol Chem ; 378(8): 837-41, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9377479

ABSTRACT

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.


Subject(s)
Crithidia fasciculata/enzymology , Electron Transport Complex IV/metabolism , Mitochondria/enzymology , Animals , Blotting, Western , Crithidia fasciculata/ultrastructure , Cyclooxygenase 2 , Cysteine/chemistry , Cysteine/metabolism , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/isolation & purification , Isoenzymes/chemistry , Isoenzymes/immunology , Peroxidases/chemistry , Peroxidases/immunology , Prostaglandin-Endoperoxide Synthases/chemistry , Prostaglandin-Endoperoxide Synthases/immunology , RNA, Protozoan/biosynthesis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
11.
Trends Microbiol ; 5(5): 189-95, 1997 May.
Article in English | MEDLINE | ID: mdl-9160507

ABSTRACT

The editing of the mitochondrial RNAs of kinetoplastid protozoa is a bizarre form of transcript maturation that involves insertion and deletion of uridylate residues. Editing leads to the formation of translational initiation and termination codons, the correction of gene-encoded reading frame shifts and the creation of complete reading frames in mRNAs. It is therefore an essential step in mitochondrial gene expression.


Subject(s)
Eukaryota/genetics , Eukaryota/metabolism , RNA Processing, Post-Transcriptional , RNA/metabolism , Animals , Gene Expression Regulation , Mutagenesis, Insertional , RNA, Mitochondrial , Sequence Deletion , Transcription, Genetic , Uridine/genetics
12.
Mol Biochem Parasitol ; 85(2): 171-86, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9106191

ABSTRACT

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.


Subject(s)
Crithidia fasciculata/chemistry , Crithidia fasciculata/genetics , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Crithidia fasciculata/enzymology , Dicyclohexylcarbodiimide/chemistry , Dicyclohexylcarbodiimide/metabolism , Electron Transport/genetics , Electrophoresis, Gel, Two-Dimensional , Molecular Sequence Data , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Sequence Analysis
13.
Mol Biochem Parasitol ; 79(1): 47-59, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8844671

ABSTRACT

Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of CuA in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C. fasciculata cytochrome c oxidase consists of at least 10 different subunits. N-terminal sequences were obtained from the six smallest subunits of the complex, one of them showing significant similarity to Neurospora crassa cytochrome c oxidase subunit V. The N-terminus of each of the four largest subunits was found to be blocked.


Subject(s)
Crithidia fasciculata/enzymology , Electron Transport Complex IV/isolation & purification , Amino Acid Sequence , Animals , Electron Spin Resonance Spectroscopy , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Insecta/parasitology , Kinetics , Molecular Sequence Data , Protein Conformation , Sequence Analysis , Sequence Homology, Amino Acid , Spectrophotometry
14.
J Clin Invest ; 98(2): 358-64, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8755645

ABSTRACT

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence. Three sisters were shown to suffer from fish-eye disease (FED), a disorder which is caused by mutations in the gene coding for lecithin:cholesterol acyltransferase (LCAT). Sequencing of the LCAT gene of all three probands revealed compound heterozygosity for a missense mutation in exon 4 which is reported to underlie the FED phenotype, and a point mutation located in intron 4 (IVS4:T-22C). By performing in vitro expression of LCAT minigenes and reverse transcriptase PCR on mRNA isolated from leukocytes of the patient, this gene defect was shown to cause a null allele as the result of complete intron retention. In conclusion, we demonstrated that a point mutation in a lariat branchpoint consensus sequence causes a null allele in a patient with FED. In addition, our finding illustrates the importance of this sequence for normal human mRNA processing. Finally, this report provides a widely applicable strategy which ensures fast and effective screening for intronic defects that underlie differential gene expression.


Subject(s)
Corneal Diseases/genetics , Introns , Phosphatidylcholine-Sterol O-Acyltransferase/genetics , Point Mutation , Amino Acid Sequence , Base Sequence , Conserved Sequence , Corneal Diseases/enzymology , DNA Primers , Exons , Female , Genetic Carrier Screening , Humans , Male , Middle Aged , Molecular Sequence Data , Pedigree , Phosphatidylcholine-Sterol O-Acyltransferase/biosynthesis , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Polymerase Chain Reaction , RNA Precursors/metabolism , RNA, Messenger/biosynthesis
16.
Curr Opin Genet Dev ; 6(2): 221-31, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8722180

ABSTRACT

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.


Subject(s)
RNA Editing/genetics , Animals , Apolipoproteins B/genetics , Base Sequence , Mitochondria/genetics , Molecular Sequence Data , Nucleoside Deaminases/physiology , RNA/genetics , RNA, Mitochondrial , Receptors, Glutamate/genetics
18.
FEBS Lett ; 381(1-2): 123-6, 1996 Feb 26.
Article in English | MEDLINE | ID: mdl-8641419

ABSTRACT

The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA). The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile. Elements that are conserved in the C. fasciculata sequence include (i) an N-terminal (D/E)-(K/R)-X-K-(X2)-W-(X2)-(I/L) motif, (ii) a putative membrane-spanning region in the middle portion of the protein, and (iii) a C-terminal W-(X13)-(N/D)-P motif. The C. fasciculata protein is synthesized with a cleavable presequence.


Subject(s)
Crithidia fasciculata/enzymology , Electron Transport Complex IV/biosynthesis , Electron Transport Complex IV/chemistry , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA Primers , DNA, Complementary , Electron Transport Complex IV/isolation & purification , Electrophoresis, Polyacrylamide Gel , Macromolecular Substances , Mammals , Mice , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
19.
Mol Biochem Parasitol ; 73(1-2): 211-22, 1995 Jul.
Article in English | MEDLINE | ID: mdl-8577329

ABSTRACT

Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part. Chimeras formed with a specific gRNA:pre-mRNA pair displayed a large variation in length, due to variably sized 3' end truncations of the gRNA moieties and variation in the sites in the pre-mRNA to which the gRNAs were attached. Surprisingly, the presence of a U-tail in the gRNA for a large part determined the specificity of the linkage. In 60% of the cases gRNAs possessing a U-tail of at least one residue were attached to an editing site, whereas 75% of the gRNAs without Us were attached to non-editing sites. Furthermore, the chimera forming activity was greatly stimulated by the addition of ATP but not by AMP-CPP, an ATP-analogue with a non-hydrolyzable alpha-beta phosphate bond. This suggests the involvement in the chimera formation of an RNA ligase.


Subject(s)
Crithidia fasciculata/metabolism , RNA, Guide, Kinetoplastida/metabolism , RNA, Messenger/metabolism , RNA, Protozoan/metabolism , Adenosine Triphosphate/pharmacology , Animals , Base Sequence , Chimera , Cloning, Molecular , Crithidia fasciculata/genetics , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Kinetoplast/genetics , Mitochondria/metabolism , Molecular Sequence Data , NADH Dehydrogenase/genetics , RNA Editing , RNA Ligase (ATP)/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Messenger/genetics , RNA, Protozoan/genetics
20.
Eur J Biochem ; 227(3): 780-6, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7867638

ABSTRACT

RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3' oligo(U) extension. Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to mitochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3'-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The possible involvement of the proteins in the editing process is discussed.


Subject(s)
Carrier Proteins/metabolism , Crithidia fasciculata/metabolism , Oligoribonucleotides/metabolism , Protozoan Proteins/metabolism , Uracil Nucleotides/metabolism , Animals , Binding, Competitive , Carrier Proteins/genetics , Carrier Proteins/radiation effects , Crithidia fasciculata/genetics , Cross-Linking Reagents , Insecta/parasitology , Mitochondria/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/radiation effects , RNA Editing , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Species Specificity , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Ultraviolet Rays
SELECTION OF CITATIONS
SEARCH DETAIL
...