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At least 775 records · Page 43Linked to original sources
PubMed1994

Subunit 6 of the Fo-ATP synthase complex from cytoplasmic male-sterile radish: RNA editing and NH2-terminal protein sequencing.

RNA editing and NH2-terminal processing of subunit 6 (atp6) of the mitochondrial Fo-ATPase complex has been investigated for the normal (fertile) and Ogura (male-sterile) radish cytoplasms to determine if previously identified differences between the Ogura atp6 locus and its normal radish counterpart are associated with cytoplasmic male sterility. Analysis of cDNA clones from five different sterile and fertile radish lines identified one C-to-U transition, which results in the replacement of a proline with a serine, in several of the lines. No editing of atp6 transcripts was observed in two lines, Scarlet Knight (normal radish) and sterile CrGC15 (Ogura radish). This is the first example of a naturally occurring plant mitochondrial gene that is not edited. The Ogura atp6 polypeptide is synthesized with a predicted NH2-terminal extension of 174 amino acids in contrast to the nine amino acid extension found in normal radish. In spite of the lack of similarity between the two extensions, NH2-terminal sequence analysis indicates that both polypeptides are processed to yield identical core proteins with a serine as the NH2-terminal residue. These results indicate that ATPase subunit 6 is synthesized normally in Ogura radish, and that it is unlikely that the atp6 locus is associated with Ogura cytoplasmic male sterility.

Amino Acid Sequence↗
PubMed1983

The primary and secondary structure of the 5'-end region of encephalomyocarditis virus RNA. A novel approach to sequencing long RNA molecules.

The primary structure of a 149-nucleotide fragment of encephalomyocarditis (EMC) virus RNA from the 5'-terminus of the genome up to the poly(C) tract (S fragment) has been determined. For isolation of the S fragment, site-directed fragmentation of the viral RNA with RNase H and poly(dG) was employed. For sequencing the S fragment, a novel approach has been developed, which can be used for primary structure determination of long RNA molecules. A model of the secondary structure of the S fragment is proposed, according to which this region of RNA is highly structured. The role of complementary oligonucleotide stretches near both termini of the RNA molecule is discussed.

Base Sequence↗
PubMed1980

Double-stranded sequences in RNA of Drosophila melanogaster: relation to mobile dispersed genes.

Double-stranded RNA (dsRNA) sequences were isolated from either total RNA or cytoplasmic poly(A)+RNA of D. melanogaster culture cells using a method described previously /1,2/. Virtually all dsRNA was found to be of high molecular weight (> 200 base pairs) and unable to snap back after RNA melting. Thus, it corresponds to one of dsRNA classes found in mouse cells, namely, to dsRNA-A /3/. Three different cloned DNA fragments of D melanogaster which hybridized to melted dsRNa were selected among 100 randomly taken. All of them efficiently bound poly(A)+RNA and high percentage of total cellular DNA. According to these and other properties, they were assigned to a group of mobile dispersed genes of D. melanogaster. DsRNA hybridizes to all subfragments of the two mobile dispersed genes tested (mdg 1 and mdg 3). Thus, complete transcripts of mobile dispersed genes are present in dsRNA. In total RNA, transcripts from one strand are more abundant than those from another one. DsRNA is heavily enriched in the transcripts from mobile dispersed genes as compared to total or poly(A)+RNA of the cytoplasm. It has been suggested that dsRNA in D. melanogaster is formed as the result of symmetric transcription of mobile dispersed genes. At least in the cytoplasmic fraction, two complementary strands are separated in vivo and may combine during the isolation of RNA.

Chromatography, Gel↗
PubMed1990

Sequence analysis of the leader RNA of two porcine coronaviruses: transmissible gastroenteritis virus and porcine respiratory coronavirus.

The leader RNA sequence was determined for two pig coronaviruses, transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV). Primer extension, of a synthetic oligonucleotide complementary to the 5' end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Northern blot analysis, using a synthetic oligonucleotide complementary to the leader RNA, showed that the leader RNA sequence was present on all of the subgenomic mRNA species. The porcine coronavirus leader RNA sequences were compared to each other and to published coronavirus leader RNA sequences. Sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these RNA sequences.

Amino Acid Sequence↗
PubMed1999

Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences.

Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

Alternative Splicing↗
PubMed1996

Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA.

Sequences from the 5' end of type 1 human immunodeficiency virus RNA dimerize spontaneously in vitro in a reaction thought to mimic the initial step of genomic dimerization in vivo. Dimer initiation has been proposed to occur through a "kissing-loop" interaction involving a specific RNA stem-loop element designated SL1: the RNA strands first interact by base pairing through a six-base GC-rich palindrome in the loop of SL1, whose stems then isomerize to form a longer interstrand duplex. We now report a mutational analysis aimed at defining the features of SL1 RNA sequence and secondary structure required for in vitro dimer formation. Our results confirm that mutations which destroy complementarity in the SL1 loop abolish homodimer formation, but that certain complementary loop mutants can heterodimerize. However, complementarity was not sufficient to ensure dimerization, even between GC-rich loops, implying that specific loop sequences may be needed to maintain a conformation that is competent for initial dimer contact; the central GC pair of the loop palindrome appeared critical in this regard, as did two or three A residues which normally flank the palindrome. Neither the four-base bulge normally found in the SL1 stem nor the specific sequence of the stem itself was essential for the interaction; however, the stem structure was required, because interstrand complementarity alone did not support dimer formation. Electron microscopic analysis indicated that the RNA dimers formed in vitro morphologically resembled those isolated previously from retroviral particles. These results fully support the kissing-loop model and may provide a framework for systematically manipulating genomic dimerization in type 1 human immunodeficiency virus virions.

Base Sequence↗
PubMed1982

Nucleotide sequence of tobacco mosaic virus RNA.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.

Amino Acid Sequence↗
PubMed1986

Structure of RNA polymerase II promoters. Coordinate conformational alteration of the upstream activator of the TATA- and RNA-initiation sequences under moderate torsional stress.

Different conformations of the circular DNA domains containing the intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent genes (914 base pairs) were modified by in vitro ligation in various conditions. The effect of increasing torsional stress on the conformation of the composing elements was determined by analysis of the sensitivity to the single strand-specific S1 endonuclease and it was observed that the sites of conformational alterations correspond to the positions relevant for promoter function (upstream activator sequence, TATA sequence, and RNA initiation site).

Base Sequence↗
PubMed1989

RNA editing in wheat mitochondria results in the conservation of protein sequences.

RNA editing is a process that results in the production of a messenger RNA with nucleotide sequences that differ from those of the template DNA, and provides another mechanism for modulating gene expression. The phenomenon was initially described in the mitochondria of protozoa. Here we report that RNA editing is also required for the correct expression of plant mitochondrial genes. It has previously been proposed that in plant mitochondria there is a departure from the universal genetic code, with CGG specifying tryptophan instead of arginine. This was because CGG codons are often found in plant mitochondrial genes at positions corresponding to those encoding conserved tryptophans in other organisms. We have now found, however, wheat mitochondrial gene sequences containing C residues that are edited to U residues in the corresponding mRNA sequences. In this way, CGG codons can be changed to UGG codons in the mRNA so that tryptophan may be encoded according to the universal genetic code. Furthermore, for each codon modification resulting from a C----U conversion that we studied, we found a corresponding change in the amino acid that was encoded. RNA editing in wheat mitochondria can thus maintain genetic information at the RNA level and as a result contribute to the conservation of mitochondrial protein sequences among plants.

Amino Acid Sequence↗
PubMed2000

Mutations in a conserved enteroviral RNA oligonucleotide sequence affect positive strand viral RNA synthesis.

We showed earlier that a transition mutation U234C, located within the completely conserved 5 nucleotide (nt) tract 5'-CGUUA (nt232-236) in the 5' non-translated region (NTR) of the coxsackievirus B3 (CVB3) genome, attenuated CVB3 cardiovirulence in mice. To further explore the role of the sequence, we induced two single and one double transversion mutations in the conserved 5mer in a cardiovirulent CVB3 genome. The mutated sites partially or totally reverted to parental wild-type when progeny viruses were passaged at 37 degrees C, but remained stable when transfection and subsequent passages were performed at 33.5 degrees C. Viral replication in cell culture was attenuated at 37 degrees C or 39.5 degrees C relative to replication at 33.5 degrees C. While Western blot analysis demonstrated the level of protein translation consistent with virus replication, the ratios of positive to negative strand viral RNA at 37 degrees C in murine cells demonstrated a 2-5 fold diminution from those measured at 33.5 degrees C. Mutant CVB3 strains failed to replicate productively when inoculated into mice. The biological data are consistent with an hypothesis that proposes a lesion with primary effects at the level of positive strand viral RNA synthesis that results in attenuation of viral replication at physiologic temperature.

5' Untranslated Regions↗
PubMed1999

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

Animals↗
PubMed2004

A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA.

Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.

Chromatography, High Pressure Liquid↗
PubMed1995

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Amino Acid Sequence↗
PubMed2026

Multiomics approaches reveal direct NF-κB p65 target genes in pancreatic islets during cytokine exposure and in type 1 diabetes.

Autoimmune diseases, including Type 1 diabetes (T1D), are often characterized by overactive inflammatory signaling pathways. The proinflammatory cytokine interleukin-1β (IL-1β) elicits global gene expression changes in islet β-cells which overlap with islets obtained from human donors with T1D. The direct transcriptional link between NF-κB subunit p65 and target genes involved with autoimmune events was investigated. We used a multiomics approach including bulk RNA-sequencing (RNA-Seq), single-cell RNA-sequencing (scRNA-Seq), and chromatin immunoprecipitation coupled to deep sequencing (ChIP-Seq), alongside molecular docking simulations, and transcriptional assays. Through the various experimental modalities, we identified early response genes driven by IL-1β that were differentially expressed in pancreatic islets from human T1D donors and also conserved across mouse, rat, and human tissues. ChIP-Seq revealed genes that are direct genomic targets of the NF-κB p65 transcription factor. Moreover, regions that gained RNA polymerase II binding following cellular exposure to IL-1β were identified, complementing the early response gene profile induced by β-cell exposure to IL-1β. Molecular docking simulations predicted that mutations reducing p65 transcriptional capacity do not alter DNA binding ability. These findings clearly show that IL-1β signaling in pancreatic β-cells directs p65 to specific genomic regions congruent with increased gene expression relevant to T1D in β-cell lines as well as mouse and human islets exposed to cytokines. Islets from human donors with T1D express genes identified as direct p65 targets using unbiased approaches, implicating heightened NF-κB activity as a critical component of autoimmune disease etiology.NEW & NOTEWORTHY Using multiple Seq-based approaches, this study identified genes expressed in human pancreatic tissue from donors with Type 1 diabetes that are regulated acutely by exposure to the cytokine interleukin-1beta. The NF-kB transcription factor p65 (RelA) was determined via ChIP-Seq to be a major control node regulating this immediate early response. These collective datasets are consistent with a paradigm of overactive NF-kB signaling as a critical component of autoimmunity in both rodents and humans.

Humans↗
PubMed2026

Ovarian H3K27ac remodeling is associated with impaired follicular development in laying hens with fatty liver hemorrhagic syndrome.

Fatty liver hemorrhagic syndrome (FLHS) is a metabolic disease of laying hens that reduces egg production and is accompanied by reproductive impairment, but the ovarian regulatory mechanisms that connect nutritional stress to follicular dysfunction remain unclear. This study examined whether active chromatin remodeling in the ovary is associated with FLHS induced by a high-energy, low-protein (HELP) diet. Hy-Line Brown hens were assigned to a basal diet or HELP diet, and ovarian tissue was collected for histone H3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing and RNA sequencing. The HELP diet reduced laying performance and the numbers of small yellow and hierarchical follicles, indicating compromised follicular development. Genome-wide H3K27ac profiling identified 2,111 regions with lower acetylation and 1,707 regions with higher acetylation in FLHS ovaries. Genes linked to differential H3K27ac regions were enriched in pathways related to oocyte meiosis, cell cycle control, FoxO signaling, gonadotropin-releasing hormone signaling, and steroid hormone biosynthesis. RNA sequencing identified 341 differentially expressed genes, with a predominance of downregulated genes. Integration of chromatin and transcriptome data highlighted folliculogenesis-related genes, including FGF1, FGF9, and MMP10, that showed reduced H3K27ac enrichment together with decreased expression. Super-enhancer analysis further identified 131 regions with reduced H3K27ac signal in FLHS ovaries, including regions located near PCNA and RAP1A, two genes involved in cellular proliferation and survival signaling. Motif enrichment of differential H3K27ac regions implicated Fos, SF-1/NR5A1, and GATA-4 as candidate transcriptional regulators. These findings indicate that HELP diet-induced FLHS is associated with broad attenuation of active ovarian regulatory elements and reduced expression of genes required for follicle growth, tissue remodeling, and steroidogenic function. The study provides an ovarian epigenomic framework for understanding reproductive decline in FLHS-affected laying hens.

Fatty liver hemorrhagic syndrome↗
PubMed2026

Ionizing radiation induces bidirectional transcriptomic reprogramming and dynamic NOS2/TREM2 regulation in triple-negative breast cancer cells.

PURPOSE: To characterize irradiation-associated transcriptomic changes in murine triple-negative breast cancer cells and examine dose- and time-response patterns of selected radiation-responsive candidates. MATERIALS AND METHODS: RNA sequencing (RNA-seq) was performed in 4T1 cells collected 24 h after 4 Gy irradiation, followed by Reactome and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and gene set enrichment analyses. Representative RNA-seq-derived genes were examined by reverse transcription quantitative PCR (RT-qPCR), and selected immune- and inflammation-related transcripts were further assessed across additional radiation doses and post-irradiation time points. Inducible nitric oxide synthase (NOS2) and triggering receptor expressed on myeloid cells 2 (TREM2) protein abundance was assessed by Western blotting, and nitrite accumulation in culture supernatants was measured using a Griess reagent-based assay as an indirect readout of nitric oxide production. RESULTS: RNA sequencing identified 757 differentially expressed genes, including 285 upregulated and 472 downregulated genes. Irradiation was associated with enrichment of inflammatory, interferon-related, immune-system, and cell-adhesion transcriptional signatures, whereas downregulated genes were enriched in cell-cycle-, chromosome-cohesion-, DNA-damage-response-, DNA-repair-, and SUMOylation-related pathways. Selected immune- and inflammation-related transcripts showed distinct temporal patterns. Nos2 mRNA increased across the examined 0-6 Gy dose range and at later post-irradiation time points, whereas NOS2 protein showed different kinetics, with an early peak after 4 Gy irradiation and no clear further increase above 6 Gy. Nitrite accumulation increased after irradiation. Trem2 showed the largest fold increase among strongly upregulated transcripts identified by RNA-seq, but RT-qPCR detected a significant increase only at 24 h, and TREM2 protein abundance remained unchanged across the examined doses and time points. CONCLUSIONS: Ionizing radiation was associated with broad bidirectional transcriptional remodeling in 4T1 cells, involving immune-, inflammatory-, and interferon-related signatures together with reduced representation of cell-cycle- and DNA-repair-related gene sets. The discordant mRNA and protein patterns of NOS2 and TREM2 indicate that transcript-level responses do not necessarily translate into corresponding protein-level changes. These findings define irradiation-associated molecular responses requiring further functional investigation.

Triple-negative breast cancer↗