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PubMed1995

Enterovirus 71 isolated from China is serologically similar to the prototype E71 BrCr strain but differs in the 5'-noncoding region.

Enterovirus 71 H (E71 H), an isolate from an adult patient with hand-food-mouth disease (HFMD) in China, was serologically similar to the prototype strain E71 BrCr, which was isolated from a patient with aseptic meningitis. The study further analyzed the similarity of E71 H to E71 BrCr at the 5'-noncoding region (NCR), a location in genomic RNA that recently was found to be related to neurovirulence in poliovirus and Venezuelan equine encephalitis virus. Using a reverse transcription-polymerase chain reaction (RT-PCR) technique and a unique primer pair I, a 397 bp product was detected from E71 BrCr, Cox A9 (Griggs), Cox A16 (NIH), Cox B1 (HA antigen 201-468), Cox B5 (wild type), and ECHO 11 (Gregory), but not from E71 H, Cox A24 (Joseph), and ECHO 5 (Noyce). However, all of the viruses generated a 154 bp product using a universal enterovirus primer pair II. Further comparative analysis using primer-directed sequencing of both the E71 H and E71 BrCr 154 bp products revealed that they differed by 12 bases. The variations between the two viruses were clustered in two loci, one in the region of nucleotides 43-61 with eight variations, and the other in the region of nucleotides 120-133 with three variations. The differences within the 5'-NCR between the E71 H (HFMD) and the E71 BrCr (aseptic meningitis) viruses might provide a clue to explain why E71 was associated with two different clinical patterns: polio-like disease in the United States. Australia, and Eastern Europe, HFMD in China, Japan, and Singapore.

Adult↗

PubMed1978

A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes.

Xenopus 5S RNA genes in recombinant form with the plasmid pMB9 are transcribed accurately when added to a supernatant fraction obtained from disrupted nuclei of Xenopus laevis oocytes. After an initial 30 min lag period, the rate of synthesis of 5S RNA is constant for at least an hour and synthesis is still detected after 18 hr. As much as 40% of the total RNA synthesized from the recombinant DNA used in these experiments can be 5S RNA. The coding strand of the 5S RNA genes is transcribed at a rate 10 to 15 times greater than the noncoding strand. Plasmid and spacer DNA, however, are also transcribed. What fraction of total RNA synthesized is 5S RNA is strongly affected by DNA concentration, ionic strength and MgCl2 concentration. Inhibition of transcription by intermediate concentrations of alpha-amanitin demonstrates that RNA polymerase III transcribes at least 90% of all RNA synthesized. Adenovirus 2 DNA is also transcribed in the nuclear supernatant by RNA polymerase III. Approximately 15% of the total RNA synthesized migrates in an acrylamide gel as a band of 5.5S RNA and has been identified as virus-associated RNA1 by its oligonucleotide fingerprint.

Animals↗

PubMed1991

The phosphoenolpyruvate carboxylase gene family of Sorghum: promoter structures, amino acid sequences and expression of genes.

Two different members of the phosphoenolpyruvate carboxylase(PEPC)-encoding multigene family (clones lambda CP21 and lambda CP46) have been isolated from a Sorghum vulgare lambda EMBL4 genomic library. The use of the 3'-noncoding regions to probe Northern blots of RNA from roots, etiolated leaves and green leaves indicated that lambda CP21 and lambda CP46 encode the C3- and C4-type leaf PEPC isoforms, respectively. The lambda CP21 clone is expressed in the three tissues and is not light-regulated, whereas lambda CP46 is only expressed in greening leaves. The nucleotide sequence of the 5'-flanking DNA (520 bp) has been determined for both genes. For lambda CP46, several direct repeats were located in this region with similarities to sequences found in other light-regulated genes, but not in lambda CP21. The deduced amino acid sequences of the two S. vulgare PEPC proteins are 75% identical.

Amino Acid Sequence↗

PubMed2003

MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.

Base Sequence↗

PubMed2001

Deletion or substitution of the aphthovirus 3' NCR abrogates infectivity and virus replication.

The 3' noncoding region (NCR) of the genomic picornaviral RNA is believed to contain major cis-acting signals required for negative-strand RNA synthesis. The 3' NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3' NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3' NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3' NCR is essential to complete a full replication cycle and that FMDV and SVDV 3' NCRs are not functionally interchangeable.

3' Untranslated Regions↗

PubMed1985

Primary structure of histidine-tRNA synthetase and characterization of hisS transcripts.

Histidine-tRNA synthetase is one of the smallest bacterial aminoacyl-tRNA synthetases. It is less than one-half the size of the largest aminoacyl-tRNA synthetases. The entire nucleotide sequence of the Escherichia coli hisS locus was determined. The coding region is comprised of 424 codons, and the sequence was determined for 200 nucleotides on the 5'- and 3'-sides of the coding region. The translated nucleotide sequence was confirmed extensively by independent amino acid sequence information obtained by Edman degradations of purified peptides and by measurements of peptide masses by fast atom bombardment mass spectrometry. A significant sequence alignment of four bacterial aminoacyl-tRNA synthetases was reported recently (Webster, T., Tsai, H., Kula, M., Mackie, G., and Schimmel, P. (1984) Science 226, 1315-1317). Although the four enzymes vary considerably in length, this match occurs within the first 100 amino acids of each of the four enzymes and is in the segment believed to be part of the catalytic core. But no strong alignment could be found of the histidine sequence with these four tRNA synthetase sequences. This enzyme may be derived, therefore, from a different progenitor. Previous work suggested that three places in the hisS 5'-noncoding sequence could be promoter sites for RNA polymerase (Eisenbeis, S. J., and Parker, J. (1982) Gene 18, 107-114). We detected a 1400-nucleotide RNA species by RNA blot analysis with a hisS-specific probe. S1 nuclease mapping demonstrated a 5'-end to the RNA species occurs at -67 +/- 1, relative to the first nucleotide of the coding region. This position coincides with the predicted start site for transcription from one of the previously proposed promoter sites.

Amino Acid Sequence↗

PubMed1985

Characterization of the 7S RNA and its gene from halobacteria.

The 7S RNA is an abundant nonribosomal RNA in H. halobium and other halobacteria. A specific 7S RNA gene probe shows high homology to genomic DNA of all halobacteria tested but not to those of several other archaebacteria, eubacteria and eukaryotes. All halobacterial genomes seem to carry a single copy of the 7S RNA gene. The coding region of the 7S RNA gene is highly G+C rich whereas the 5'- and 3'-noncoding regions possess a rather low G+C content. An extended double stranded structure for the 7S RNA is deduced from its nucleotide sequence. The 7S RNA of H. halobium (304 nucleotides) resembles in size and structure the 7S-L RNA from mammalian cells and shares with it a sequence homology of about 50% when arranged in a colinear fashion. The similarities in sequence are found particularly at the 3'- and 5'-termini. No similarity was detected between the 7S RNA from H. halobium and the nonribosomal 6S RNA from Escherichia coli.

Animals↗

PubMed1984

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone.

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.

Amino Acid Sequence↗

PubMed2026

An inflammatory bowel disease-linked lncRNA suppresses transcription factor T-BET expression in T cells to limit intestinal inflammation.

Among the tens of thousands of annotated long noncoding RNAs (lncRNAs) in the human genome, only a small fraction have been functionally characterized. Here, we show that a well-established inflammatory bowel disease (IBD) risk locus encoded a conserved lncRNA, lnc15 (2310015A10Rik/ENSMUSG00000097729), whose structure was destabilized by risk-associated variants, leading to its degradation. Deletion of lnc15 in mice resulted in molecular features of inflammation under steady-state conditions and conferred heightened susceptibility to experimental colitis. Lnc15 was abundantly expressed in T cells, with highest expression in regulatory T (Treg) cells. Mechanistically, lnc15 suppressed the transcription factor T-BET by recruiting the CCR4-NOT RNA degradation complex to Tbx21 mRNA. Our study identifies that lnc15 simultaneously enhances Treg cell suppressive function and impairs conventional T cell pathogenicity in the context of intestinal inflammation. Collectively, these findings identify lnc15 as a functional lncRNA that links noncoding genetic variation to immune regulation and prevention of mucosal inflammation. VIDEO ABSTRACT.

RNA, Long Noncoding↗

PubMed1994

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.

Base Sequence↗

PubMed2000

Sequence analysis and genome organisation of poinsettia mosaic virus (PnMV) reveal closer relationship to marafiviruses than to tymoviruses.

Sequence comparison and genome organisation of poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, revealed a closer relationship to marafiviruses. The complete nucleotide sequence of PnMV was determined. The 6099-nt RNA genome encodes a putative 221-kDa polyprotein that lacks a stop codon between the replicase and the coat protein genes, as in most tymovirus RNAs. The genomic RNA has a poly(A) tail at its 3'-terminus in contrast to the tRNA-like structure found in the RNA of most tymoviruses, and no homology was observed to the conserved noncoding region of the tymoviral 3'-termini. The tymobox of PnMV, a 16-nt region of the subgenomic RNA (sgRNA) promoter shared by most tymoviruses, differs in 3 nt from the RNA sequence of tymoviruses but is identical to the sequence of marafiviruses. At least three sgRNAs were found in PnMV-infected Euphorbia pulcherrima and in isolated PnMV particles; one that is 650 nt long encodes the 21.4-kDa coat protein, and the others are about 3.5 and 1.7 kb and contain the 5'- and the 3'-terminal parts of genomic RNA, respectively. Like tymoviruses, PnMV particles sediment as top and bottom components. The particles of the top component contain the sgRNA (650 nt) encoding the coat protein, and those of bottom component contain both genomic and sgRNAs.

Amino Acid Sequence↗

PubMed2005

A surprisingly large RNase P RNA in Candida glabrata.

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

Ascomycota↗

PubMed1991

The 5' noncoding sequences from a less virulent Theiler's virus dramatically attenuate GDVII neurovirulence.

RNA transcripts derived from recombinant chimeras between the highly virulent GDVII virus and the less virulent BeAn virus were constructed to study the molecular pathogenesis of Theiler's murine encephalomyelitis virus infection. The presence of the BeAn 5' noncoding sequences in chimera 2 (BeAn 5' noncoding sequences joined with the GDVII nucleotides encoding the polyprotein and present in the 3' end) resulted in dramatic attenuation of GDVII neurovirulence and development of poliomyelitis in mice. This reduced neurovirulence was associated with slower virus growth and lower peak titers in the brain and spinal cord than with parental GDVII virus replication. On the other hand, the sites of replication following chimera 2 infection were the same as those seen in GDVII-infected mice; the distribution of virus antigen and histopathological changes indicated that chimera 2 replicates in neurons in the brain, e.g., in the neocortex, hippocampus, caudate putamen, and brain stem, as well as in anterior-horn cells in the spinal cord. Chimera 2 was efficiently cleared from the mouse central nervous system by day 30 postinfection, in marked contrast to the persistence of the BeAn parent in the central nervous system. This suggests that elements in the BeAn sequences that encode the polyprotein or are present in the 3' noncoding region are necessary for viral persistence. It is of interest that chimera 2-infected mice developed localized inflammatory, demyelinating lesions which were detected at day 28 postinfection but these lesions did not become larger with time. Thus, virus persistence appears to be required for maintenance and progression of immune-mediated demyelination. If the demyelinating lesions become sufficiently large, clinical signs and disease may develop.

Acute Disease↗

PubMed2006

Distinctive signatures of histone methylation in transcribed coding and noncoding human beta-globin sequences.

The establishment of epigenetic marks, such as methylation on histone tails, is mechanistically linked to RNA polymerase II within active genes. To explore the interplay between these modifications in transcribed noncoding as well as coding sequences, we analyzed epigenetic modification and chromatin structure at high resolution across 300 kb of human chromosome 11, including the beta-globin locus which is extensively transcribed in intergenic regions. Monomethylated H3K4, K9, and K36 were broadly distributed, while hypermethylated forms appeared to different extents across the region in a manner reflecting transcriptional activity. The trimethylation of H3K4 and H3K9 correlated within the most highly transcribed sequences. The H3K36me3 mark was more broadly detected in transcribed coding and noncoding sequences, suggesting that K36me3 is a stable mark on sequences transcribed at any level. Most epigenetic and chromatin structural features did not undergo transitions at the presumed borders of the globin domain where the insulator factor CTCF interacts, raising questions about the function of the borders.

Acetylation↗

PubMed1997

Chronic variant of myocarditis associated with hepatitis C virus infection.

BACKGROUND: Although molecular biological studies suggest a pathogenic link between enterovirus infection and dilated cardiomyopathy (DCM), the frequency of detection of enteroviral RNA is not consistently high in myocardial tissue from patients with DCM. A recent study has suggested that hepatitis C virus (HCV) may also be involved in the development of DCM. METHODS AND RESULTS: We performed genomic analysis for HCV in three patients with chronic active myocarditis. In all three patients, serum aminotransferase activities remained within normal ranges until the terminal stage of heart failure. At necropsy, all three livers showed evidence of tissue damage caused by chronic congestion, and one liver had evidence of chronic hepatitis. Routinely processed, paraffin-embedded tissue blocks of myocardium and liver were analyzed. Renal specimens were also analyzed to exclude the possibility of myocardial contamination with HCV material from the circulating blood. RNA extracted from the heart, liver, and kidney was subjected to strand-specific reverse transcription and amplified by semi-nested polymerase chain reaction. The target nucleotide sequence was a 178-bp fragment of the highly conserved 5'-noncoding region. Both positive- (genomic) and negative-strand RNA (replicative intermediates) were present in myocardial and liver tissue samples from all three patients. However, negative-strand RNA was undetectable in renal tissue from one patient. CONCLUSIONS: These findings suggest that HCV replicated in myocardial tissue of these patients with myocarditis. Thus, HCV infection may contribute to the development of this unusual form of myocarditis.

Chronic Disease↗

PubMed1987

Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.

Amino Acid Sequence↗

PubMed1989

The origin of polynucleotide-directed protein synthesis.

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that, at first, the simple attachment of amino acids to the 2'(3')-termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Adenosine Monophosphate↗

PubMed2004

Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs.

RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1). Here we show that Rdp1 is associated with two conserved proteins, Hrr1, an RNA helicase, and Cid12, a member of the polyA polymerase family, in a complex that has RNA-directed RNA polymerase activity (RDRC, RNA-directed RNA polymerase complex). RDRC physically interacts with RITS in a manner that requires the Dicer ribonuclease (Dcr1) and the Clr4 histone methyltransferase. Moreover, both complexes are localized to the nucleus and associate with noncoding centromeric RNAs in a Dcr1-dependent manner. In cells lacking Rdp1, Hrr1, or Cid12, RITS complexes are devoid of siRNAs and fail to localize to centromeric DNA repeats to initiate heterochromatin assembly. These findings reveal a physical and functional link between Rdp1 and RITS and suggest that noncoding RNAs provide a platform for siRNA-dependent localization of RNAi complexes to specific chromosome regions.

Adenosine Triphosphatases↗