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PubMed2026

Pathogenic human mitochondrial tRNA variants impair RNA processing by compromising 5' leader removal.

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

Journal Article↗

PubMed1986

Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.

Amino Acid Sequence↗

PubMed1998

Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus strain Beaudette C and completion of the entire genome sequence.

The nucleotide sequences of the nucleocapsid protein (NP) gene, the intergenic regions in the nucleocapsid protein (NP)-phosphoprotein (P), P-matrix protein (M) and M-fusion glycoprotein gene junctions and the trailer region of a virulent Newcastle disease virus (NDV) strain Beaudette C were determined. The NP gene is 1747 nt long and encodes a protein of 489 amino acids. Each of the intergenic sequences determined is 1 nt long and, including the previously published intergenic sequences, the gene junction sequences varied in length from 1-47 nt and lacked any sequence identity. The 5' trailer region is 113 nt in length. Comparison of the sequences of the terminal leader and trailer regions of Beaudette C strain with those of nonvirulent strain B1 showed a high level of conservation, indicating the likelihood of these elements not being a factor in virulence. Together with previously published data, this report completes the sequence of the 15,186 nt genomic RNA of NDV strain Beaudette C.

Base Sequence↗

PubMed1985

Yeast contains two functional genes coding for ribosomal protein S10.

The DNA sequence of the second copy of the gene coding for yeast ribosomal protein S10 was determined and compared with the sequence of the first gene-copy. In addition, the sites at which the transcription of these genes start and terminate are identified. The amino acid coding regions of the two gene copies are virtually identical. The leader and in particular the trailer sequences, however, are significantly different, while the intervening sequences have hardly any homology. Taking advantage of the sequence differences we could establish that both genes are expressed in the vegetatively growing yeast cell; the respective transcripts, however, differ in their relative amounts.

Amino Acid Sequence↗

PubMed2005

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus.

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3' leader-N-4a(P)-4b-M-G-L-5' trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Amino Acid Motifs↗

PubMed1987

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme.

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2001

This is the end: processing, editing and repair at the tRNA 3'-terminus.

The generation of a mature tRNA 3'-end is an important step in the processing pathways leading to functional tRNA molecules. While 5'-end processing by RNase P is similar in all organisms, generation of the mature 3'-terminus seems to be more variable and complex. The first step in this reaction is the removal of 3'-trailer sequences. In bacteria, this is a multistep process performed by endo- and exonucleases. In contrast, the majority of eukaryotes generate the mature tRNA 3'-end in a single step reaction, which consists of an endonucleolytic cut at the tRNA terminus. After removal of the 3'-trailer, a terminal CCA triplet has to be added to allow charging of the tRNA with its cognate amino acid. The enzyme catalyzing this reaction is tRNA nucleotidyltransferase, homologs of which have been found in representatives of all three kingdoms. Furthermore, in metazoan mitochondria, some genes encode 3'-terminally truncated tRNAs, which are restored in an editing reaction in order to yield functional tRNAs. Interestingly, this reaction is not restricted to distinct tRNAs, but seems to act on a variety of tRNA molecules and represents therefore a more general tRNA repair mechanism than a specialized editing reaction. In this review, the current knowledge about these crucial reactions is summarized.

Animals↗

PubMed1984

The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem.

We have cloned and sequenced the wild-type and suppressor alleles of the S. pombe sup8 tRNA gene. The wild-type allele has a leucine UAA anticodon and the suppressor (sup8-e) carries the opal suppressor anticodon UCA. The gene has a 16 base pair intervening sequence that, in the RNA, is predicted to form a secondary structure which involves base pairing to the 5', rather than the usual 3' side of the 5' splice site. When incubated in Saccharomyces cerevisiae cell-free extracts both alleles are efficiently transcribed, the 5' leader and 3' trailer sequences are removed and CCA is added to the 3' processed end; however, the intervening sequence is not excised. This finding implies that the structural requirements of the splicing endonucleases in the two yeasts have diverged. No other tRNA genes with related sequences were detected in S. pombe DNA by hybridization, suggesting that other UUA isoacceptors may be structurally dissimilar to sup8 or that the UUA codon may be decoded by a UUG leucine isoacceptor.

Alleles↗

PubMed1992

Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae.

A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions. Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA. Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined. Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required. Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase. Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure. The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step. Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr). A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced. These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.

Base Sequence↗

PubMed1990

The intron of the yeast actin gene contains the promoter for an antisense RNA.

Using Northern blot analysis we have detected an approximately 840 nucleotide-long RNA which is complementary to the 5' leader sequence and the first ten nucleotides of the coding sequence of the yeast actin (ACT1) messenger RNA. We have determined two transcription start sites for this actin antisense RNA (ASR1), both within the ACT1 intron, at about 80 and 90 nucleotides downstream from the 5' splice site. Analysis of a cDNA clone showed that this RNA species overlaps the entire trailer sequence and approximately 20 nucleotides of the coding sequence of the nearby yeast YPT1 gene.

Actins↗

PubMed1999

RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs.

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Arabidopsis↗

PubMed2005

Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda.

The entire genomic RNA of the Gulu (Uganda 2000) strain of Ebola virus was sequenced and compared to the genomes of other filoviruses. This data represents the first comprehensive genetic analysis for a representative isolate of the Sudan species of Ebola virus. The genome organization of the Sudan species is nearly identical to that of the Zaire species, but the presence of a gene overlap (between GP and VP30 genes) and a longer trailer sequence distinguish it from that of the Reston species. As has been observed with other filoviruses, stemloop structures were predicted to form at the 5' end of Ebola Sudan mRNA molecules, and the genomic RNA termini showed a high degree of sequence complimentarity. Comparisons of the amino acid sequences of encoded gene products shows that there is a comparable level of identity or similarity between Ebola virus species, with Sudan and Zaire actually showing a slightly closer relationship to the Reston species than to one another. These comparisons also indicated that the VP24 is the most conserved Ebola virus protein (followed closely by the VP40 and L proteins), while the GP is the least conserved gene product. The most divergent regions were seen in the C-terminus of GP1 (mucin-like region) and within the C-terminal third of the nucleoprotein sequence.

5' Untranslated Regions↗

PubMed1986

Structural organization and sequence of the homeotic gene Antennapedia of Drosophila melanogaster.

The structure of the Drosophila melanogaster Antennapedia (Antp) gene has been investigated by the isolation and sequencing of different cDNAs and genomic clones. Northern analysis, S1 mapping and primer extension experiments reveal a complex and unusual gene structure. The gene is composed of two promoters, eight exons spanning >100 kb, and two termination processing regions. Four major polyadenylated transcripts were found, two of them starting at a second internal promoter in front of exon 3. All four transcripts have extremely long untranslated leader and trailer sequences in the range of 1-2 kb. Despite the complex transcriptional organization, the open reading frame is the same in all transcripts, and starts in exon 5 giving rise to a protein of mol. wt. 42 800. The putative protein is rich in glutamine (18%) and proline (10%). The homeobox, a region which previously has been shown to be highly conserved among homeotic genes, is contained in the open reading frame and located in the last exon. Functional implications of the complex structure with respect to development and its relation to the mutant phenotypes are discussed.

Amino Acid Sequence↗

PubMed2001

Complete nucleotide sequences of Nipah virus isolates from Malaysia.

We have completely sequenced the genomes of two Nipah virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 18246 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the Hendra virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily.

Base Sequence↗

PubMed1995

Upstream promoter elements are sufficient for selenocysteine tRNA[Ser]Sec gene transcription and to determine the transcription start point.

The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA[Ser]Sec gene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3'-trailer sequence. Replacement of the tRNA[Ser]Sec coding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA[Ser]Sec promoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA[Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point (tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp.

Animals↗

PubMed2004

Identification of viral genomic elements responsible for rabies virus neuroinvasiveness.

Attenuated tissue culture-adapted and natural street rabies virus (RV) strains differ greatly in their neuroinvasiveness. To identify the elements responsible for the ability of an RV to enter the CNS from a peripheral site and to cause lethal neurological disease, we constructed a full-length cDNA clone of silver-haired bat-associated RV (SHBRV) strain 18 and exchanged the genes encoding RV proteins and genomic sequences of this highly neuroinvasive RV strain with those of a highly attenuated nonneuroinvasive RV vaccine strain (SN0). Analysis of the recombinant RV (SB0), which was recovered from SHBRV-18 cDNA, indicated that this RV is phenotypically indistinguishable from WT SHBRV-18. Characterization of the chimeric viruses revealed that in addition to the RV glycoprotein, which plays a predominant role in the ability of an RV to invade the CNS from a peripheral site, viral elements such as the trailer sequence, the RV polymerase, and the pseudogene contribute to RV neuroinvasiveness. Analyses also revealed that neuroinvasiveness of an RV correlates inversely with the time necessary for internalization of RV virions and with the capacity of the virus to grow in neuroblastoma cells.

Animals↗

PubMed1991

Characterization of the memory gene dunce of Drosophila melanogaster.

The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases or base-pairs). To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs. Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes. Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites: two of these were mapped by parallel S1-nuclease and primer extension experiments. In addition, some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3' ends. The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3' splice sites separated by six base-pairs. Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation. The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1981

Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors.

A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences.

Animals↗