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Results for “Direct RNA sequencing”

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PubMed1994

Unusual heterogeneity of leader-mRNA fusion in a murine coronavirus: implications for the mechanism of RNA transcription and recombination.

Coronavirus mRNA transcription was thought to be regulated by the interaction between the leader RNA and the intergenic sequence (IS), probably involving direct RNA-RNA interactions between complementary sequences. In this study, we found that a particular strain of mouse hepatitis virus, JHM2c, which has a deletion of a 9-nucleotide (nt) sequence (UUUAUAAAC) immediately downstream of the leader RNA, transcribed subgenomic mRNA species containing a whole array of heterogeneous leader fusion sites. Using a transfected defective interfering RNA which contains an IS and a reporter (chloramphenicol acetyltransferase) gene and JHM2c as a helper virus, we demonstrated that subgenomic mRNAs transcribed from the defective interfering RNAs were extremely heterogeneous. The leader-mRNA fusion sites in this virus can be grouped into five types. In type I, the leader is fused with the consensus IS of the template RNA at a site within the UCUAA repeats, consistent with the classical model of discontinuous transcription. In type II, the leader is fused with the consensus IS as in type I, but the leader of mRNA contains some nucleotide substitutions within the UCUAA repeats. In type III, the leader is fused with mRNAs at a site either upstream or downstream of the consensus IS. The sequences around the fusion sites bear little or no homology to the leader. As a result, mRNAs contain sequences complementary to the template sequences upstream of the IS or have sequence deletions downstream of the IS. In type IV, the leader is fused to the IS at the 9-nt sequence immediately downstream of the UCUAA repeats. In type V, the leader-mRNA fusion site contains a duplication of a portion of the leader sequence or an insertion of nontemplated sequences which are not present in either leader or template RNA. These patterns of leader-mRNA fusion resemble the aberrant homologous recombination frequently seen in other RNA viruses. The degree of heterogeneity of leader fusion sites is dependent on the sequences of both the leader RNA and IS. These results suggest that leader-mRNA fusion in coronavirus transcription does not require direct RNA-RNA interaction between complementary sequences. A modified model of RNA transcription and recombination based on protein-RNA and protein-protein interactions is proposed. This study also provides a paradigm for aberrant homologous recombination.

Animals↗

PubMed1971

Hybridization properties of DNA sequences directing the synthesis of messenger RNA and heterogeneous nuclear RNA.

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D(o)t (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D(o)t about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11 degrees C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4 degrees -6 degrees C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.

Animals↗

PubMed1990

The Euglena gracilis chloroplast rpoB gene. Novel gene organization and transcription of the RNA polymerase subunit operon.

The rpoB gene coding for a beta-like subunit of the chloroplast DNA-dependent RNA polymerase has been located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. We have determined 5760 base-pairs of DNA sequence, including 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resembles the E. coli rpoB-rpoC and higher plant chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with a predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp respectively and a Group II intron of 309 bp. All other known rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNA, as well as rpoB-rpoC1 and rpoB1-rpoC1-rpoC2 mRNAs were identified.

Amino Acid Sequence↗

PubMed2026

Dogme: a nextflow pipeline for reprocessing nanopore RNA and DNA modifications.

MOTIVATION: Oxford Nanopore (ONT) sequencing allows for the direct detection of RNA and DNA modifications from unamplified nucleic acids, which is a significant advantage over other platforms. However, the rapid updates to ONT basecalling models and the evolving landscape of computational tools for modification detection bring about challenges for reproducible and standardized analyses. To address these challenges, we developed Dogme to automate basecalling, alignment, modification detection, and transcript quantification. Dogme automates the reprocessing of ONT POD5 files by integrating basecalling using Dorado, read mapping using minimap2 and subsequent analysis steps such as running modkit. The pipeline supports three major types of sequencing data-direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA). Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2'-O-methylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA. RESULTS: We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected 96 603 m6A, 43 476 m5C, 8829 inosine, 10 055 pseudouridine, and 30 320 Nm sites in three biological replicates. The pipeline produced reproducible modification profiles and transcript expression levels across replicates, demonstrating its utility for integrative long-read transcriptomic and epigenomic analyses. AVAILABILITY AND IMPLEMENTATION: Dogme is implemented in Nextflow and is freely available under the MIT license at https://github.com/mortazavilab/dogme, with documentation provided for installation and usage.

RNA↗

PubMed1986

Nucleotide sequence at the 3' end of Japanese encephalitis virus genomic RNA.

Japanese encephalitis (JE) virus genomic RNAs were purified from virions. Two hundred nucleotides at the 3' end of JE virus genomic RNA were directly sequenced by using reverse transcriptase. The nucleotide sequence at the 3' end of the viral RNA was conserved among four kinds of JE virus strains. The sequence has no AU-rich region that is present at the 3' termini of alphavirus RNAs. We also compared the nucleotide sequences at the 3' ends of RNAs from three different flaviviruses and found several common sequence elements. A secondary structure at the 3' end of JE virus genomic RNA was proposed that may be common among flavivirus genomic RNAs. Such structures and other common stretches of nucleotide sequences may be related to the biological properties of flaviviruses.

Base Sequence↗

PubMed1988

EIAV genomic organization: further characterization by sequencing of purified glycoproteins and cDNA.

Nucleotide sequence analyses of two different proviral clones of equine infectious anemia virus (EIAV), designated lambda 12 (K. Rushlow et al., 1986, Virology 155, 309-321) and 1369 (T. Kawakami et al., 1987, Virology 158, 300-312), indicate significant differences in the organization of two critical regions of the viral genome, i.e., in the short open reading frames in the pol-env intergenic region and in the 5'-end of the env gene. To determine the correct structure of the EIAV genome, we have performed nucleotide sequence analyses of cDNA clones produced from viral RNA and direct sequencing of purified EIAV envelope glycoproteins (gp90 and gp45). The results of the cDNA sequencing confirm the presence of two short open reading frames in the pol-env intergenic region, as reported previously for the lambda 12 clone. The protein sequencing data correlated exactly with the amino-terminal sequences of gp90 and gp45 deduced from lambda 12 nucleotide sequences. However, the protein sequencing also revealed that the putative signal sequence of EIAV gp90 is not removed during processing. Thus, EIAV apparently contains short open reading frames analogous to human immunodeficiency virus, but differs in its mode of env polyprotein processing.

Amino Acid Sequence↗

PubMed1991

Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending.

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.

Animals↗

PubMed1991

Apolipoprotein B mRNA editing is an intranuclear event that occurs posttranscriptionally coincident with splicing and polyadenylation.

The subcellular compartment in which apolipoprotein (apo) B mRNA is edited is unknown. We studied the site of endogenous apoB mRNA editing and correlated the extent of editing with mRNA maturation in the rat liver. RNA editing activity was demonstrated in both nuclear and cytoplasmic extracts. The specific activity of the editing activity was 5.5-fold higher in the nuclear extract, which was not accounted for by activators, inhibitors, or modulators. However, the total editing activity was 3.1 times higher in the cytoplasmic extract. Highly purified rat liver nuclear apoB mRNA contained 17.3 +/- 1.45% edited sequences compared with 56 +/- 2.5% and 62.15 +/- 6.2% edited sequences in hepatic total and polysomal RNAs, respectively. Because of the significant extent of editing of total nuclear RNA, we fractionated it into a poly(A-) and poly(A+) fraction. While the poly(A-) nuclear fraction contained only 10.4 +/- 1.1% edited sequences, which represents a maximum estimate, the poly(A+) nuclear apoB mRNA contained 50 +/- 1.8% edited sequences, a value very similar to that for polysomal RNA. By direct sequencing of cDNA and genomic clones, we found that as in the case of the human apoB gene, the rat apoB gene contains an intron 25 immediately upstream of the edited exon 26. Using this information, we developed a method to examine in a highly selective manner apoB mRNA that is present in the nucleus before splicing of intron 25 and after splicing of this intron. The unspliced nuclear pre-mRNA contained 7.4 +/- 0.2% edited sequences compared with 51.0 +/- 0.9% edited sequences in the spliced nuclear apoB mRNA. Furthermore, in the poly(A-) pool of apoB pre-mRNA, unspliced nuclear pre-mRNA contained hardly any (1.56%) edited sequences, and the spliced nuclear pre-mRNA contained 7.8 +/- 0.6% edited mRNA. In the poly(A+) fraction, unspliced nuclear pre-mRNA had 25.4 +/- 0.05% and spliced nuclear mRNA 53 +/- 0.6% of its apoB mRNA in an edited form. We conclude that in the rat liver apoB mRNA editing is not a cotransciptional event. It occurs posttranscriptionally, but the process is essentially complete in the spliced polyadenylated apoB mRNA before it leaves the nucleus. Little, if any, additional editing occurs in the cytoplasmic compartment.

Animals↗

PubMed1995

Requirements for the self-directed replication of flock house virus RNA 1.

The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Animals↗

PubMed1995

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗

PubMed1994

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.

Actins↗

PubMed1991

Sequence organization and RNA structural motifs directing the mouse primary rRNA-processing event.

The first processing step in the maturation of mouse precursor rRNA involves cleavage at nucleotide ca. +650, at the 5' border of a 200-nucleotide region that is conserved across mammals and contains the sequences that direct the processing. To identify the relevant sequence elements, we used rRNAs with small internal mutations and short pre-rRNA substrates. Much of the region can be mutated without appreciable effect, but nucleotides +655 to +666 appear to be absolutely required and short segments surrounding +750 and +810 markedly stimulate processing. The minimal processing signal corresponds to rRNA nucleotides +645 to +672. Formation of a ribonucleoprotein complex of retarded electrophoretic mobility is evidently necessary but not sufficient for processing. Computer-assisted analysis suggested a phylogenetic- and mutant-supported secondary structure in which the minimal processing signal forms a stem with the +655 region in the loop, and there is a separate branched duplex containing the downstream stimulatory sequences. Use of antisense RNA, in trans and in cis, to sequester the +655 region in a duplex supported the hypothesis that this critical region was needed in a single-stranded conformation for processing and for specific complex formation.

Animals↗

PubMed1992

Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.

Contained within a single cell, the fertilized egg, is information that will ultimately specify the entire organism. During early embryonic cleavages, cells acquire distinct fates and their differences in developmental potential might be explained by localization of informational molecules in the egg. The mechanisms by which Vg1 RNA, a maternal mRNA, is translocated to the vegetal pole of Xenopus oocytes may indicate how developmental signals are localized. Data presented here show that a 340-nucleotide localization signal present in the 3' untranslated region of Vg1 RNA is sufficient to direct RNA localization to the vegetal pole.

Animals↗

PubMed1993

The nucleotide sequence of a satellite RNA associated with strawberry latent ringspot virus.

The nucleotide sequence of a satellite RNA associated with a strawberry isolate (H) of strawberry latent ringspot nepovirus (SLRSV) was determined from cDNA copies and the 5' end sequence was deduced from directly sequenced virion RNA. At the 3' end a poly(A) sequence was identified. A long open reading frame encoding a polypeptide of 331 amino acids (M(r) 36488) was determined. Sequence comparisons showed that SLRSV satellite RNA has no extensive homology with other sequences in the GenEmbl and Swiss-Prot databases.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of the original Brazilian isolate of coleus yellow viroid from Solenostemon scutellarioides and infectivity of its complementary DNA.

The complete nucleotide (nt) sequence of the original coleus yellow viroid (CYVd) from Solenostemon scutellarioides, 'Golden Bedder', has been determined. The covalently closed single-stranded CYVd RNA molecule consists of 248 nt residues which assumes a rod-like secondary structure when folded in the model of lowest free energy. The sequence was determined by direct sequencing of RNA and from three overlapping cDNA clones. Comparison of the CYVd sequence with that of Coleus blumei viroid 1 (CbVd 1) from Germany demonstrated that they are closely related. The differences observed in the genome organization of CYVd relative to CbVd 1 were at three sites: position 25 (one U deletion), position 26 (a U was replaced by an A) and position 241 (one A insertion). The first two mutations were detected in one A-rich segment of eight nt (between positions 25 and 34). Northern blot hybridization of partially purified nucleic acids from the leaf tissue of S. scutellarioides 'Frilled Fantasy', inoculated with double-stranded cDNA, demonstrated that this fragment was infectious. These data enable CYVd to be assigned to the viroid class of plant pathogens, based on its biological properties and molecular structure. This work also gives additional support to the present classification system, in which the viroids isolated from S. scutellarioides form a distinct subgroup.

Base Sequence↗

PubMed1989

The genome structure of turnip crinkle virus.

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.

Amino Acid Sequence↗

PubMed1981

The leftward promoter of bacteriophage lambda. Structure, biological activity, and influence by adjacent regions.

The effect of regions adjacent to the lambda PL promoter was studied using a sequence deleted in an A/T-rich segment immediately upstream from the promoter. High resolution thermal denaturation analysis showed that the undeleted sequence, as isolated on a 360-bp restriction fragment (360-PL) melted in two distinct steps. Since the deleted sequence (230-PL) melts at a higher temperature than any portion of 360-PL, the deleted promoter is more stable to denaturation. This increased stability was also suggested by a transcription assay in which the strong binding of RNA polymerase is inhibited at low temperature. The deleted promoter fragment required a higher temperature to become functional than did the 360-PL fragment. Furthermore, RNA polymerase bound to the 230-PL fragment is displaced by heparin at a faster rate than from 360-PL, and the deleted fragment initiates transcription at a reduced rate. Thus, by several criteria the deleted promoter is less active than the parent sequence. Evidence is presented arguing that the deletion does not alter the sequence directly recognized by RNA polymerase. Therefore, the altered sequence is reducing promoter activity by changing some other level of overall promoter structure. These results suggest that regions outside of the sequence directly recognized by a regulatory protein can affect genetic expression from the site.

Bacteriophage lambda↗

PubMed1990

Glucagon gene 3'-flanking sequences direct formation of proglucagon messenger RNA 3'-ends in islet and nonislet cells lines.

Glucagon and the glucagon-like peptides are encoded within a larger precursor, proglucagon. Transcription of the glucagon gene in pancreas, intestine, and brain gives rise to identical proglucagon mRNA transcripts, after which tissue-specific post-translational processing produces different profiles of proglucagon-derived peptides in each tissue. The importance of glucagon gene 3'-untranslated and 3'-flanking sequences in the control of glucagon mRNA production was studied by transfecting a series of 3'-deleted glucagon genes into fibroblast and islet cell lines. Glucagon genes containing 2 kilobases of 3'-flanking sequences gave rise to accurately processed mRNA transcripts in both baby hamster kidney fibroblasts and InR1-G9 islet cell lines. Deletion of all but 50 basepairs of 3'-flanking sequence had no effect on glucagon mRNA 3'-end formation. In contrast, additional deletion of 3'-flanking and 3'-untranslated sequences resulted in the production of read-through mRNA transcripts with aberrant 3'-ends. The results of these studies define a 50-basepair region in the 3'-flanking sequence of the glucagon gene important for the accurate processing of proglucagon mRNA transcripts.

Animals↗