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At least 1,189 records · Page 66Linked to original sources
PubMed2002

Characterization of a viroid-derived RNA promoter for the DNA-dependent RNA polymerase from Escherichia coli.

This paper attributes a novel function, namely, that of transcriptional promoter, to the self-complementary, self-cleaving hammerhead RNA sequences found in RNA derived from the peach latent mosaic viroid (PLMVd). The features of this RNA promoter, which adopts a hairpin structure that can be utilized by Escherichia coli RNA polymerase (RNAP) for in vitro transcription, that trigger the RNAP driven transcription and are responsible for the specific initiation of synthesis are described. The essential requirement for initiation is a basepaired uridine adjacent to the loop. The presence of a loop composed of at least six nucleotides connected to a relatively unstable stem significantly increases the level of initiation. Finally, we present several insights into the mechanism of the RNAP which reveal that it behaves differently with an RNA template as compared to a DNA one.

Base Sequence↗
PubMed1997

Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.

Animals↗
PubMed2026

Trimethylamine-producing microbe Bacillus megaterium KCTC 3007 promotes antitumor immunity in endometrial cancer via type I interferon response pathways.

BACKGROUND: Endometrial cancer (ECa) is one of the most common gynecologic malignancies, with limited therapeutic responses in metastatic or recurrent cases. The bacterial microbiota has emerged as a key modulator of carcinogenesis and antitumor immunity. However, the role of endometrial microbiota in ECa pathogenesis and prognosis remains poorly understood. METHODS: We performed comprehensive multi-omics analysis integrating metatranscriptomics, transcriptomics, and targeted metabolomics from 60 ECa and 18 benign patients. RNA sequencing enabled simultaneous profiling of active tissue-resident microbiota and host gene expression. Serum metabolomics was conducted on all patients. Identified microbial-metabolite associations were validated through in vitro co-culture experiments using peripheral blood mononuclear cells (PBMCs), cancer cell lines, RNA sequencing, and live cell imaging. RESULTS: ECa patients exhibited significantly altered microbial diversity and composition compared to benign controls. Through integrated multi-omics analysis, we identified Bacillus megaterium (BM) KCTC 3007 as a beneficial microbe associated with prolonged recurrence-free survival. In an exploratory analysis of ECa subtypes, Cupriavidus taiwanensis and Marinomonas primoryensis showed potential links to poor prognosis, although these observations warrant caution due to the limited size of certain subgroups. Tissue BM abundance positively correlated with serum trimethylamine N-oxide (TMAO) levels, particularly in postmenopausal women. In vitro experiments demonstrated that BM KCTC 3007 enhanced antitumor immunity by promoting interleukin and type I interferon expression, expanding CD8 + T cell populations, and increasing immune cell-tumor cell interactions. RNA sequencing revealed activation of interferon alpha response and immune cell proliferation pathways, with IFNAR1 identified as a key upstream regulator. TMAO treatment recapitulated these immune-activating effects, enhancing CD8 + T cell responses and preferentially inducing pyroptotic cancer cell death. CONCLUSIONS: We provide the first evidence that tissue-resident BM KCTC 3007 promotes antitumor immunity in ECa through TMAO production and subsequent type I interferon-mediated immune activation. This integrated multi-omics approach establishes a complete microbe-metabolite-host mechanistic pathway and highlights the therapeutic potential of TMAO-producing probiotic strains for ECa treatment. Video Abstract.

Female↗
PubMed2000

Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient.

AIMS: To ascertain the clinical relevance of a strain of Enterobacteriaceae isolated from the stool of a bone marrow transplant recipient with diarrhoea. The isolate could not be identified to the genus level by conventional phenotypic methods and required 16S ribosomal RNA (rRNA) gene sequencing for full identification. METHODS: The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GNI+) and API (20E) systems. Genotypically, the 16S bacterial rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by multiple sequence alignment. RESULTS: Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The isolate was identified as Salmonella arizonae (73%) and Escherichia coli (76%) by the Vitek (GNI+) and API (20E) systems, respectively. 16S rRNA sequencing showed that there was only one base difference between the isolate and E coli K-12, but 48 and 47 base differences between the isolate and S typhimurium (NCTC 8391) and S typhi (St111), respectively, showing that it was an E coli strain. The patient did not require any specific treatment and the diarrhoea subsided spontaneously. CONCLUSIONS: 16S rRNA gene sequencing was useful in ascertaining the clinical relevance of the strain of Enterobacteriaceae isolated from the stool of the bone marrow transplant recipient with diarrhoea.

Adult↗
PubMed1980

Directed deletion of a yeast transfer RNA intervening sequence.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

Base Sequence↗
PubMed1993

Small subunit ribosomal RNA gene sequence of the oyster parasite Perkinsus marinus.

The small subunit rRNA gene of the oyster pathogen Perkinsus marinus was characterized from cells of infected oyster hemolymph by polymerase chain reaction and molecular cloning. The gene, 1,793 nucleotides in size, has 77.2% sequence similarity to that of its host, the eastern oyster Crassostrea virginica. The sequence was confirmed using recently available in vitro cultures of P. marinus. DNA from pure P. marinus culture was amplified with specific primers synthesized according to the sequence from infected oyster hemolymph, and predicted size fragments were obtained. Furthermore, restriction digests yielded fragments of expected size in amplified rDNA from in vitro cultures. The P. marinus sequence has 97.5% similarity to the Perkinsus sp. sequence from the Australian mollusc Anadara trapezia.

Animals↗
PubMed2001

The massively parallel genetic algorithm for RNA folding: MIMD implementation and population variation.

A massively parallel Genetic Algorithm (GA) has been applied to RNA sequence folding on three different computer architectures. The GA, an evolution-like algorithm that is applied to a large population of RNA structures based on a pool of helical stems derived from an RNA sequence, evolves this population in parallel. The algorithm was originally designed and developed for a 16384 processor SIMD (Single Instruction Multiple Data) MasPar MP-2. More recently it has been adapted to a 64 processor MIMD (Multiple Instruction Multiple Data) SGI ORIGIN 2000, and a 512 processor MIMD CRAY T3E. The MIMD version of the algorithm raises issues concerning RNA structure data-layout and processor communication. In addition, the effects of population variation on the predicted results are discussed. Also presented are the scaling properties of the algorithm from the perspective of the number of physical processors utilized and the number of virtual processors (RNA structures) operated upon.

Algorithms↗
PubMed1998

Orthologous DNA sequence variation among 5S ribosomal RNA gene spacer sequences on homoeologous chromosomes 1B, 1D, and 1R of wheat and rye.

5S ribosomal gene spacer sequences from the short-spacer arrays of wheat and rye were isolated by PCR. The 29 new DNA sequences displayed noticeable heterogeneity at scattered positions. Nevertheless, based on shared DNA sequence polymorphisms, sequence alignment clearly classified the sequences into three groups. Group-specific primer sets were designed to allow chromosomal assignment by PCR on nullitetrasomic wheat stocks, as well as on wheat-rye translocation and addition lines. The three groups were assigned to orthologous loci 5S-Rrna-B1, 5S-Rrna-D1, and 5S-Rrna-R1 on homoeologous chromosomes 1B, 1D, and 1R, respectively. Hence, group-specific DNA sequence variation could be related to fixed orthologous DNA sequence variation between 5S rRNA multigene families on the homoeologous group 1 chromosomes. In addition, members of the three groups showed fixed orthologous length polymorphism. Four sequenced 5S-Rrna-B1 units, however, had a duplication in the gene encoding region and are probably representatives of a nontranscribed subfamily of 5S rDNA repeating units. The observed chromosome-specific polymorphisms among sequences belonging to a multigene family with thousands of copies suggests that this type of polymorphism may exist in many genes and gene families in polyploid wheats. The implication of this finding in relation to the construction of molecular tools for wheat-genome analysis and manipulation is discussed.

Base Sequence↗
PubMed1990

Complex transcription from the extrachromosomal DNA encoding mitochondrial functions of Plasmodium yoelii.

All tested members of genus Plasmodium contain tandemly arrayed, transcribed, extrachromosomal DNA with a unit length of 6.0 kb. This DNA contains two open reading frames with potential to encode cytochrome c oxidase subunit I (cox1) and cytochrome b (cob) as well as fragments of rRNA genes scattered on both strands. At least 10 discrete RNA molecules transcribed during erythrocytic stages of a rodent malarial parasite, Plasmodium yoelii, were recognized by the 6.0-kb DNA probes. The RNA molecules of 1.4 and 1.1 kb were identified as encoding cox1 and cob, respectively. Primer extension and RNA sequencing were used to locate and characterize 5' ends of these two RNAs, showing that an identical 12-nucleotide sequence, 5'-TATTTTT TGTTT-3', was present at these positions. This sequence may act as a promoter or as an RNA processing signal. A stem-loop structure signifying a possible transcription termination was present at the end of the cox1 open reading frame. At least six discrete RNA molecules of less than 250 nucleotides were recognized by different fractions of the 6.0-kb DNA. The largest of these, 200 nucleotides, was also characterized by primer extension and RNA sequencing. This molecule had a high homology to portions of the large-subunit rRNA domains IV and V. Other, small RNA molecules were recognized by regions of the 6.0-kb DNA that had homology to the highly conserved peptidyltransferase domain of large-subunit rRNA. These results show that the unusual compactly organized mitochondrionlike DNA of malarial parasites is transcribed in a complex pattern.

Amino Acid Sequence↗
PubMed1995

Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses.

The genome of cocksfoot mottle virus (CfMV) is a positive-sense ssRNA molecule of 4082 nucleotides as revealed by sequencing the entire genome. The 5'-untranslated region of the genome is 69 nucleotides and the 3'-untranslated region is 225 nucleotides in length. The coding region contains four open reading frames (ORFs). The organization of CfMV ORFs differs significantly from that of the previously sequenced sobemoviruses southern bean mosaic virus and rice yellow mottle virus. ORF1 encodes a protein having a calculated molecular mass of 12.3 kDa. The function of this protein is unknown. The next ORF codes for the putative VPg and serine protease. The ORF2a product consists of 568 amino acids, with a calculated molecular mass of 60.9 kDa. The replicase of CfMV is translated as part of a polyprotein by--1 ribosomal frameshifting in ORF2a. The calculated molecular mass of the transframe protein is 103.4 kDa. ORF3 encodes the 27.6 kDa coat protein. This has been verified by amino acid sequencing of the CfMV coat protein N terminus. Northern blots of total RNA from CfMV-infected barley leaves reveal the 4.1 kb genomic RNA band and one virus-specific band of 1.2 kb, which may represent a subgenomic RNA for coat protein synthesis.

Amino Acid Sequence↗
PubMed2001

The RNA/protein symmetry hypothesis: experimental support for reverse translation of primitive proteins.

Although the "RNA-world" theory, or the RNA-first theory is renowned for a promising theory of biogenesis, it is also possible that both RNAs and proteins have coevolved forming a stable metabolic complex from the very beginning. I investigated this possibility assuming that the genetic information flowed symmetrically in the era of the origin of life, i.e. the primitive translation machinery worked in both directions (from RNA to protein and from protein to RNA). According to this RNA/protein symmetry theory, the genetic information would have come from existing cellular proteins via reverse translation. This process would have been completed in a short period of time without searching an enormous RNA sequence space. Furthermore, reverse translation would have ensured biological continuity; proteins that were essential for cellular metabolism would have been utilized in the same way as before the protein sequence information would have been transferred into the RNA sequences. I also propose a possible mechanism for the process of reverse translation. The reverse translation would proceed in the 3' to 5' direction using a set of at least 20 reverse transfer RNAs (rtRNAs) that can recognize their specific amino acid residue and carry their corresponding codon. A source of genetic information would be a primary sequence of a protein molecule. Several basic steps of reverse translation were demonstrated using rtRNA(Arg).

3' Untranslated Regions↗
PubMed1999

A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities.

An important group of splicing factors involved in constitutive and alternative splicing contain an arginine/serine (RS)-rich domain. We have previously demonstrated the existence of such factors in plants and report now on a new family of splicing factors (termed the RSZ family) from Arabidopsis thaliana which additionally harbor a Zn knuckle motif similar to the human splicing factor 9G8. Although only around 20 kDa in size, members of this family possess a multi-domain structure. In addition to the N-terminal RNA recognition motif (RRM), a Zn finger motif of the CCHC-type is inserted in an RGG-rich region; all three motifs are known to contribute to RNA binding. The C-terminal domain has a characteristic repeated structure which is very arginine-rich and centered around an SP dipeptide. One member of this family, atRSZp22, has been shown to be a phosphoprotein with properties similar to SR proteins. Furthermore, atRSZp22 was able to complement efficiently splicing deficient mammalian S100 as well as h9G8-depleted extracts. RNA binding assays to selected RNA sequences indicate an RNA binding specificity similar to the human splicing factors 9G8 and SRp20. Taken together, these result show that atRSZp22 is a true plant splicing factor which combines structural and functional features of both h9G8 and hSRp20.

Alternative Splicing↗
PubMed1980

Comparison of substrate base sequences for RNA ligase reactions in the synthesis of a tetradecanucleotide corresponding to bases 21-34 of E. coli tRNAfMet 1.

A tetradecanucleotide U-A-G-C(U-C-G)2G-G-C-Up corresponding to bases 21-34 of a nascent sequence of formylmethionyl tRNA of E. coli has been synthesized by the joining of two combinations of chemically synthesized oligonucleotides: 1) U-A-G-C + U-C-G-U-C-G + G-G-C-Up and 2) U-A-G-C + U-C-G-U + C-G-G-G-C-Up. In reaction 1) and the extent of joining *pG-G-C-Up to U-C-G-U-C-G was only 15.4% and the last ligation of the decamer to U-A-G-U proceeded to 27%. In reaction 2) joining between U-A-G-C and pU-C-G-Up gave a high yield (88%). The ligation of this octamer and *pC-G-G-G-C-Up also gave a satisfactory yield (52%). These reactions suggest that sequence preferences in RNA ligase reactions may arise from the structure of the 3'-end of acceptor molecules.

Base Sequence↗
PubMed1999

Ribonuclease-resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus.

BACKGROUND: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences. METHODS: A consensus 412-base sequence from the 5'NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat. RESULTS: AR-HCV-2b was fully recoverable from human plasma incubated at 4 degrees C for >300 days. The particles were tested in three clinical assay formats: Amplicor HCV Monitor 1.0, Quantiplex HCV RNA 2.0, and INNO-LiPA HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay. CONCLUSION: The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.

Base Sequence↗
PubMed2026

Phenotypic and transcriptomic characterization of biallelic RNU2-2 developmental and epileptic encephalopathy.

OBJECTIVE: A significant proportion of individuals with suspected genetic developmental and epileptic encephalopathies (DEEs) remain unsolved following whole genome sequencing (WGS). Here we describe biallelic RNU2-2 variants causing a recently reported, severe, recessive DEE. METHODS: We screened individuals who have received WGS analyses at the Genomic Medicine Centre Karolinska for Rare Diseases for biallelic RNU2-2 variants. Deep phenotyping was performed through reviewing entire medical histories and phenotypic traits were transcribed to their corresponding Human Phenotype Ontology (HPO) term. HPO terms were used to generate pairwise phenotypic similarity scores and assess for significantly shared phenotype enrichment in the RNU2-2 sub-cohort. RNA sequencing analyses were performed in fibroblast and blood tissues to compare splicing events between RNU2-2 individuals and two independent control groups. RESULTS: We identified 14 individuals from nine families with 12 ultra-rare biallelic RNU2-2 variants clustering in the conserved 5' domains. Genotype data from 13 of 14 individuals has been reported previously as part of a larger cohort. All individuals presented with a highly concordant, severe DEE, characterized by severe to profound intellectual disability, inability to walk or communicate, hyperkinesia, and refractory seizures. Infantile spasms and tonic seizures were the predominant seizure types and a Lennox-Gastaut syndrome-like phenotype was common. These individuals had a significantly similar phenotypic signature when compared with 703 individuals with complex pediatric epilepsies (two-sided Monte Carlo permutation test, p = .005). RNA sequencing analyses showed aberrant splicing, with the most pronounced effects in fibroblast tissues in mutually exclusive exon and alternate 3' splice-site events, which were not detectable in blood. SIGNIFICANCE: We present deep phenotyping data and transcriptomic analyses that provide support for rare, 5' clustering biallelic RNU2-2 variants causing this novel, severe DEE. We propose an RNA sequencing methodology on fibroblast tissue for future validation of RNU2-2 variants.

autosomal recessive disease↗
PubMed2002

High-affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1).

Long interspersed nuclear element 1 (LINE-1 or L1) is an interspersed repeated DNA found in mammalian genomes. L1 achieved its high copy number by retrotransposition, a process that requires the two L1-encoded proteins, ORF1p and ORF2p. The role of ORF1p in the retrotransposition cycle is incompletely understood, but it is known to bind single-stranded nucleic acids and act as a nucleic acid chaperone. This study assesses the nature and specificity of the interaction of ORF1p with RNA. Results of coimmunoprecipitation experiments demonstrate that ORF1p preferentially binds a single T1 nuclease digestion product of 38 nucleotides (nt) within the full-length mouse L1 transcript. The 38-nt fragment is localized within L1 RNA and found to be sufficient for binding by ORF1p but not necessary, because its complement is also efficiently coimmunoprecipitated, as are all sequences 38 nt or longer. Results of nitrocellulose filter-binding assays demonstrate that the binding of ORF1p to RNA does not require divalent cations but is sensitive to the concentration of monovalent cation. Both sense and antisense transcripts bind with apparent K(D)s in the low nanomolar range. The results of both types of assay unambiguously support the conclusion that purified ORF1p from mouse L1 is a high-affinity, non-sequence-specific RNA binding protein.

Animals↗
PubMed1978

Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.

Adenosine Triphosphate↗