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PubMed1989

An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus.

The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.

DNA↗
PubMed1987

Widespread transcription of a Qa region gene in adult mice.

The mouse MHC class I family includes genes encoded in four regions: H-2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene.

Animals↗
PubMed1990

A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology.

A deletion of 30 bases was created in the noncoding region of the human immunodeficiency virus type 1 genome that extends between the 5' splice donor and the start of the gag gene. Viral particles produced after transfection of this mutant provirus had a normal protein pattern but a reduced RNA content. The infectivity of the mutant virus was also markedly reduced but not completely abolished. Electron-microscopic examination of the mutant virions revealed major abnormalities of the nucleoid structure, mostly related to the dense material characteristic of mature particles, suggesting that the presence of RNA is essential to the normal structure of the nucleoid.

Base Sequence↗
PubMed1990

Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism.

The authentic initiation site on encephalomyocarditis virus (strain R) RNA is the 11th AUG codon (at nt. 834) from the 5' end of the viral RNA, the downstream of the two AUGs in the sequence....ACGAUGAUAAUAUGGCC... In order to assess the role of ribosome scanning in the selection of the correct initiation site, transcripts of a construct comprising the viral 5' noncoding sequence fused to a reporter gene were translated in vitro and the relative frequency of initiation at these two AUG codons, AUG-10 and AUG-11, was assayed. When deletions from the 5' end were made that retained less than 100 nucleotides of the viral 5'-noncoding sequence, initiation was highly cap-dependent and AUG-10 was utilized in preference to AUG-11, consistent with the scanning ribosome model. On the other hand, when the constructs included the entire 574 nt. segment of the 5' noncoding region situated between the poly(C) tract of the virion RNA and nt. 834, initiation was cap-independent and occurred exclusively at AUG-11, with negligible use of AUG-10. These results suggest that structural features of the 574 nucleotide segment cause initiating ribosomes to bind directly to AUG-11, without scanning the immediate upstream sequences that include AUG-10.

Base Sequence↗
PubMed1982

Contact points between a positive transcription factor and the Xenopus 5S RNA gene.

We have investigated the contact points of a positive transcription factor with the internal control region of the 5S ribosomal RNA genes of Xenopus. The methylation of any one of eight G residues clustered at the 3' end of the internal control region on the noncoding strand of the DNA or the ethylation of their surrounding phosphates interferes with the binding of this protein. The importance of the 5' half of the control region is shown by a binding affinity of the protein to oocyte 5S RNA genes that is one fourth that to somatic 5S RNA genes. This can be attributed to two base changes at residues 53 and 55 in the 5' part of the control region. The different affinities of the transcription factor for the oocyte and somatic 5S RNA genes may contribute to their differential expression in somatic cells. The almost exclusive location of strong contact points on the noncoding strand of the gene suggests how transcription events could proceed repeatedly along a gene that is assembled into a stable transcription complex. It is proposed that the complex is transiently shifted to the noncoding strand as each round of RNA synthesis occurs from the coding strand.

Animals↗
PubMed2002

[Amplification of hepatitis C virus 5' untranslated region gene by RACE and its secondary structure analysis].

OBJECTIVE: To obtain very end full-length cDNA of hepatitis C virus (HCV) 5' untranslated region (5' UTR), and analyse its primary and secondary structure. METHODS: By reverse transcription-nested polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP), a patient infected with genotype 2a HCV was found. Total RNA isolated from the serum as template, the cDNA of 5' noncoding region was amplified using rapid amplification of cDNA ends methods (RACE), the fragments were recombined by A-T clone strategy, the recombinants were confirmed by RFLP and PCR then sequenced. Secondary structures were analysed by RNA draw. RESULTS: Very end full-length cDNA of 2a genotype HCV 5' UTR was obtained by RACE. In five clones obtained, three contained full-length 5' UTR cDNA, and A21G, G170A, T222C, T247C, C339T substitutions were found compared with HC-J6. he homologies with HCV-1,HC-J6,HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93.0%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions did not alter the secondary structure. Two out of five clones were deleted to have 53 and 144 bases at 5' terminus of HCV 5' UTR, respectively. CONCLUSIONS: RACE is rapid and effective, works well to obtain very end of virus genome. With that, Authors obtained full-length cDNA of genotype 2a of HCV 5' UTR. There are genes deleted at 5' terminus circulated in hepatitis C patients.

5' Untranslated Regions↗
PubMed2005

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent.

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

Animals↗
PubMed1982

cDNA recombinant plasmid complementary to mRNAs for light chains 1 and 3 of mouse skeletal muscle myosin.

A recombinant plasmid with a cDNA sequence transcribed from mouse skeletal muscle RNA is shown to hybridize with mRNAs for myosin light chains LC1F and LC3F. The inserted fragment corresponds exclusively to the 3'-noncoding region of the mRNA. It hybridizes almost exclusively with the two light chain messengers from fast skeletal muscle RNA of adult mouse. Slight hybridization is seen with RNA from heart muscle and embryonic skeletal muscle. The implications of the conservation of the 3'-noncoding regions between the two mRNAs are discussed.

Animals↗
PubMed2025

Widespread induction of SINE-RNA expression in the mouse brain following transient focal ischemia.

Ischemic stroke triggers rapid transcriptional changes in the brain, including the induction of noncoding RNAs, which are well-established regulators of post-stroke pathophysiology. Among the numerous classes of noncoding RNAs, short interspersed nuclear element RNAs (SINE-RNAs) are transcribed by Pol III and reported to be upregulated in various paradigms of cellular stress. In the ischemic brain, Pol III-driven gene expression is not well-studied and the expression of SINE-RNAs is virtually unmapped. In the current study, we used a mouse model of transient focal ischemia to evaluate for the first time post-stroke SINE-RNA expression in the cerebral cortex on a genome-wide scale. We observed SINE-RNA induction as early as 0 to 3 h of reperfusion and peak expression at 6 h of reperfusion, with 335 SINE-RNAs induced at this timepoint as compared to sham controls. Many of these transcripts remained induced through later timepoints during the acute phase of reperfusion (24 h). Fluorescence in situ hybridization against the SINE-RNAs, combined with immunohistochemistry for cell-type markers, revealed that these RNAs are localized to the nuclei of post-ischemic neurons and microglia in the ipsilateral cortex and hippocampus in both males and females. Further, we found that SINE-RNA expression was recapitulated in vitro following oxygen-glucose deprivation in HT22 hippocampal neurons, showing that they are reproducibly expressed in neurons in both in vivo and in vitro models of ischemia. Together, this is the first study to map genome-wide SINE-RNA expression in the post-ischemic brain and reveals a new layer of the noncoding transcriptome that may play a role in the post-stroke pathophysiology.

Animals↗
PubMed2001

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs.

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.

Animals↗
PubMed1985

A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure.

The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

Base Sequence↗
PubMed1984

RNase H-catalyzed site-specific deadenylylation of rabbit alpha- and beta- globin mRNAs. Secondary structure of 3'-noncoding regions.

A general method is described for the removal of 3'-terminal polyadenylate tracts from eukaryotic messenger RNA to generate essentially homogeneous length products that can be 3'-end labeled and sequenced. Hybridization of specific oligodeoxyribonucleotides was used to direct ribonuclease H to the junction of the 3'-noncoding region and the polyadenylate sequence of rabbit alpha and beta globin mRNAs. Site-specific deadenylylation of both globin mRNAs is demonstrated by partial enzymatic sequence analysis following 3'-terminal labeling with 5'-pCp (where p indicates the labeled phosphate group). The secondary structure of the 3'-noncoding region is studied by enzymatic digestion with S1 nuclease and cobra venom ribonuclease. Structural features of the 3'-noncoding regions of these mRNAs are described, including the protein synthesis termination and the poly(A) addition recognition sites.

Animals↗
PubMed1990

Attenuation of Mengo virus through genetic engineering of the 5' noncoding poly(C) tract.

The murine cardioviruses, such as the Mengo and encephalomyocarditis viruses, and the bovine aphthoviruses, such as foot-and-mouth disease virus, are distinguished among positive-strand RNA viruses by the presence of long homopolymeric poly(C) tracts within their 5' noncoding sequences. Although the specific lengths (60-350 bases) and sequence discontinuities (for example, uridine residues) that sometimes disrupt the homopolymer have served to characterize natural viral isolates, the biological function of the poly(C) region has never been clear. We now report that complementary DNA-mediated truncation of the Mengo virus poly(C) tract dramatically attenuates the pathogenicity of the virus in mice. Animals injected with viruses with short tracts not only survived inoculation of up to 50 micrograms live virus (10(11) plaque-forming units) but consistently produced high titres of neutralizing antibodies, which conferred long-term immunogenic protection from (normally) lethal virus challenge. We propose that analogous synthetic strains of foot and mouth disease virus could serve as the basis for new attenuated vaccines.

Animals↗
PubMed2003

Prevalence of GBV-C infection among dental personnel.

Healthcare workers who carry out exposure-prone procedures are theoretically at increased risk of acquiring blood-borne virus infections. GB virus C (GBV-C) is a recently described blood-borne virus that is related distantly to hepatitis C virus. The occupational risk of GBV-C infection to healthcare workers is unknown. This study collected detailed occupational and personal risk data in parallel with a blood specimen, to establish the prevalence and determinants of GBV-C infection among dental healthcare workers. The presence of GBV-C antibodies was detected using commercially available ELISA; GBV-C RNA was detected by nested PCR using primers from the conserved 5' noncoding region. The overall prevalence of GBV-C antibodies among the study population was 11.1% (98/880, 95% confidence interval [CI], 9.1-13.4%) and 4.6% were positive for GBV-C RNA (46/879, 95% CI, 2.5-5.1%), resulting in a cumulative prevalence of 15.7%. These figures are similar to those described in other populations. There was no significant difference in lifetime exposure to GBV-C between dentists (17.7%) and dental nurses/hygienists (14.3%). Significantly more dental nurses/hygienists aged 16-30 years had been exposed to GBV-C compared to dentists of the same age (chi(2) = 13.75; P < 0.001). Conversely, significantly more dentists 46 years or older had evidence of exposure to GBV-C compared to dental nurses/hygienists (chi(2) = 6.79; P = 0.009). The high prevalence of GBV-C infection did not seem to be related to past parenteral exposure, and the data suggest that sexual transmission, rather than occupational transmission, was a more important route for GBV-C infection among this population.

Adolescent↗
PubMed1993

Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion.

Tpl-2 is a gene encoding a protein kinase which is primarily expressed in normal spleen, thymus, and lung tissue and is activated by provirus insertion in Moloney murine leukemia virus-induced T-cell lymphomas during the late stages of oncogenesis. Tpl-2 is composed of eight exons and spans a 35-kb genomic DNA region. The provirus integrates reproducibly in the last intron and in the same transcriptional orientation as the Tpl-2 gene. This genetic change leads to the expression of enhanced steady-state levels of a truncated Tpl-2 RNA transcript which is predicted to encode a protein with an altered C-terminal domain. Tpl-2 is transcribed from two alternating promoters, P1 and P2. The RNA transcripts originating in the two promoters harbor different 5' untranslated regions derived from the alternate noncoding exons IA and IB. Utilization of the P2 promoter, which gives rise to exon IB containing Tpl-2 RNA transcripts, was detected primarily in tumor cells. The Tpl-2 protein was expressed in COS-1 cells as an N-terminal fusion with a 12-amino-acid hemagglutinin tag. Immunoprecipitation of transfected COS-1 cell lysates with antihemagglutinin or anti-Tpl-2 antibodies, followed by incubation with [gamma-32P]ATP, confirmed that Tpl-2 possesses protein kinase activity.

Animals↗
PubMed1994

cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides.

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica. A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5'-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Base Sequence↗
PubMed1998

Hepatitis G virus infection in screened Chinese blood donors.

BACKGROUND AND OBJECTIVES: To determine the prevalence of the recently identified hepatitis G virus (HGV)/GBV-C in screened Chinese paid blood donors. MATERIALS AND METHODS: Two hundred and seventy-nine plasma samples were tested for HGV RNA by RT-PCR with nested primers from the 5'-noncoding region of GBV-C. All samples were obtained from plasma or blood bags that had been screened twice by routine selection tests (ALT, HBsAg, Anti-HCV, anti-HIV, and syphilis) and were available for clinical use. RESULTS: HGV RNA was detected in 2 (4%) of 50 paid plasma donors from the Beijing Red Cross Blood Center, 1 (2%) of 50 paid blood donors from Taiyuan, and 9 (5%) of 179 paid blood donors from Hebei, a total HGV detection rate of 4.3% (12/279). CONCLUSIONS: Our data suggest that HGV infection is relatively frequent even in screened donors, at least in paid screened donors, although larger-scale studies are required.

Blood Donors↗