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At least 289 records · Page 16Linked to original sources

PubMed1990

Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U.

Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a1b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revertant (P3/119/70) of the vaccine (Y.V. Svitkin, S.V. Maslova, and V.I. Agol, 1985, Virology 147, 243-252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a1b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a1b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C472----U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5' noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.

Animals↗

PubMedDate not supplied

A sequence downstream of AAUAAA is required for rabbit beta-globin mRNA 3'-end formation.

The sequence AAUAAA, found 11-30 base pairs (bp) upstream of the poly(A) site of most non-histone eukaryotic messenger RNAs (mRNAs) forms an essential part of the recognition site for 3'-end processing of the primary transcript. However, the sequence AATAAA is found in transcribed regions of genes and is differentially utilized in genes containing multiple copies of the sequence within the 3'-noncoding region, suggesting that the hexanucleotide alone does not comprise a complete recognition site. Therefore, it seems likely that additional sequences are required to form a complete recognition site for 3'-end formation. We have investigated the sequence requirements for mRNA 3'-end formation using the rabbit beta-globin gene as a model system. Here we demonstrate that an additional sequence 3' to AAUAAA is required for the correct 3'-end formation of rabbit beta-globin mRNA.

Animals↗

PubMed1981

Sequence and gene organization of mouse mitochondrial DNA.

The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5' ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.

Animals↗

PubMed1991

An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells.

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.

Base Sequence↗

PubMed2003

A uniform system for microRNA annotation.

MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAs from diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.

MicroRNAs↗

PubMed1988

Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation.

Three clones of full-length cDNA encoding human myeloperoxidase were isolated from a human leukemia HL-60 cell cDNA library in lambda gt10 and characterized. Analysis of the nucleotide sequence of one of the cDNA clones, lambda MP-H17, indicated that the cDNA contained 3207 bp with an open reading frame of 2238 bp, a 5' noncoding region of 159 bp, a 3' noncoding region of 800 bp, and a poly(A) tail of 10 bp. cDNA of the two other clones, lambda MP-H7 and lambda MP-H14, each contained insertions with shorter sequences of 96 and 82 bp, respectively, on the open reading frame of lambda MP-H17 cDNA. A myeloperoxidase genomic clone was isolated, and the structure of its 5' region was determined and compared with the structures of these cDNAs. The comparison revealed that the three cDNAs were derived from myeloperoxidase mRNAs produced by alternative splicing from a transcript of the single gene. Nucleotide sequence analysis of the 3' region of the cDNAs of several clones indicated that the mRNAs were polyadenylated at five different sites. Amino acid sequence determination of the amino-terminal and carboxy-terminal portions of the myeloperoxidase light and heavy chains revealed that, during processing of a precursor polypeptide into the mature protein, the amino-terminal polypeptide, the small peptide between the light and heavy chains, and the carboxy-terminal amino acid were excised.

Amino Acid Sequence↗

PubMed1997

[Functions of the segment-specific noncoding regions of influenza virus genome RNA].

The genome of influenza A viruses consists of eight negative-strand RNA segments. These segments contain the untranslated regions (UTRs), ranging from 20 to 61 nucleotides, at their 3' and 5' ends. The UTRs are composed of the highly conserved terminal nucleotides and the segment-specific nonconserved nucleotides located adjacent the open reading frame of the viral RNAs. Utilizing the virus-like model RNAs, whose nonconserved UTRs were mutated, deleted or replaced with those of other segments, the unique features of the nonconserved UTRs have been elucidated in the steps of transcription, translation, replication and RNA packaging into virus particles. Here I summarize current understanding of the functions of the segment-specific nonconserved UTRs of virus RNA.

Genome, Viral↗

PubMed2000

Simultaneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay.

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the gag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5' noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Plus- and minus-stranded hepatitis G virus RNA in liver tissue and in peripheral blood mononuclear cells.

Hepatitis G virus (HGV), which was recently identified, is a single, plus-stranded RNA virus that is thought to replicate via minus-stranded RNA, but no information is available about the distribution of either plus- (genomic) or minus- (replicative) stranded HGV RNAs in HGV infected patients. We, therefore, tested the serum, liver tissue, and peripheral blood mononuclear cells (PBMCs) of six hepatitis patients with HGV infection for the presence of plus- and minus-stranded HGV RNA. The RT-nested PCR was used with primers derived from 5'-noncoding region of the genome. Before RT-PCR analysis, the 3'-termini of RNA specimens were chemically modified to discriminate between plus- and minus-stranded HGV RNA. Plus-stranded HGV RNA was detected in the serum and liver tissue of all six patients and in the PBMCs of five patients. Minus-stranded RNA was detected in the liver tissue of all six patients, in the serum of two patients, and in the PBMCs of one patient. In summary, the detection of minus-stranded HGV RNA in liver tissue may indicate that HGV replicates in the liver.

Flaviviridae↗

PubMed1993

Repression of the herpes simplex virus 1 alpha 4 gene by its gene product occurs within the context of the viral genome and is associated with all three identified cognate sites.

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha 4-1 proximal, alpha 4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha 4-2) of the alpha 4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha 22 gene and 3 hr later with the test viruses were tested in RNase protection assay for amounts of the chimeric gene RNA relative to amounts of alpha 22 gene RNA. We report the following: (i) Mutation of the alpha 4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha 4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha 4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha 4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha 4 mRNA relative to that of alpha 22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.

Animals↗

PubMed2000

Inhibition of translation of mRNAs containing gamma-monomethylphosphate cap structure in frog oocytes and in mammalian cells.

The gamma-monomethylphosphate cap structure is found in several eukaryotic small RNAs including nuclear U6, U6atac, 7SK, plant nucleolar U3, and rodent cytoplasmic B2 RNAs. In the case of human U6 snRNA, the 5' end sequence corresponding to nucleotides 1-25 serves as the capping signal and directs the formation of methylphosphate cap structure. In this study, we show that the U6 RNA capping signal, when introduced at the 5' end of RNAs, can efficiently direct the methylphosphate cap formation in RNAs of up to 2.7 kb long, as well as in different mRNAs. These data show that the methylphosphate capping signal functions in mRNAs having different primary sequences and different lengths. Presence of the methylphosphate cap structure on the 5' end of a luciferase mRNA with EMCV 5' noncoding region, which is translated in an IRES-dependent pathway, resulted in a 6- to 100-fold inhibition of translation compared to the same mRNA with a 5' triphosphate when microinjected into frog oocytes or expressed in mouse cells in tissue culture. Thus, conversion of the pppG structure to a methyl-pppG structure on the 5' end of an mRNA, which is translated in an IRES-dependent pathway, results in severe inhibition of translation. These data show that the 5' end motif of mRNAs plays an important role even in the IRES-mediated mRNA translation.

3T3 Cells↗

PubMed1994

The complete sequences of African horsesickness virus serotype 4 (vaccine strain) RNA segment 2 and 6 which encode outer capsid protein.

The complete sequences of RNA segment 2 and segment 6 of African horsesickness virus serotype 4 (AHSV-4) vaccine strain were determined from cDNA clones inserted into pBR 322. The RNAs of segment 2 and 6 are 3229, 1566 bp long respectively and both contain an open reading frame encoding proteins VP2 and VP5 of 1060, 505 amino acid residues. The estimated molecular weight of VP2 was 124,178 dalton and that of VP5 was 56,793 dalton. Their noncoding end sequences were 5'GTTTAA . . . and . . . ACATAC3' (segment 2), 5'GTTTAT . . . and . . . ACTTAC3' (segment 6). They were different from orbivirus characteristic terminal sequences, which were 5'GTTAAA . . . and . . . ACTTAC3'. The comparison of both sequences of AHSV-4 segment 2 and 6 with those of segment 2 and 5 of bluetongue virus (BTV) serotype 10 revealed 53% nucleotide similarity and 23% amino acid similarity (segment 2), and 58% nucleotide similarity and 46% amino acid similarity (segment 6). In the same way, the comparison of both sequences of the vaccine strain with those of the virulent strain segment 2 and segment 6 of AHSV-4 revealed 91% nucleotide and 96% amino acid similarity (segment 2), and 98% nucleotide and 98% amino acid similarity (segment 6).

African Horse Sickness Virus↗

PubMed2001

Characterization of the cis-acting elements controlling subgenomic mRNAs of citrus tristeza virus: production of positive- and negative-stranded 3'-terminal and positive-stranded 5'-terminal RNAs.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has an approximately 20-kb positive-sense RNA genome with two 5' ORFs translated from the genomic RNA and 10 3' genes expressed via nine or ten 3'-terminal subgenomic (sg) RNAs. The expression of the 3' genes appears to have properties intermediate between the smaller viruses of the "alphavirus supergroup" and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5' leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5' of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts -47 to -5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3'-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5'-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5'-terminal positive strand and both positive- and negative-stranded 3'-terminal RNAs. Therefore, theoretically CTV could produce 30-33 species of RNAs in infected cells.

Base Sequence↗

PubMed2003

Characterization and quantitation of differential Tsix transcripts: implications for Tsix function.

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

Animals↗

PubMed1996

A common structural core in the internal ribosome entry sites of picornavirus, hepatitis C virus, and pestivirus.

Cap-independent translations of viral RNAs of enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, hepatitis A and C viruses (HAV and HCV), and pestivirus are initiated by the direct binding of 40S ribosomal subunits to a cis-acting genetic element termed the internal ribosome entry site (IRES) or ribosome landing pad (RLP) in the 5' noncoding region (5'NCR). RNA higher ordered structure models for these IRES elements were derived by a combined approach using thermodynamic RNA folding, Monte Carlo simulation, and phylogenetic comparative analysis. The structural differences among the three groups of picornaviruses arise not only from point mutations, but also from the addition or deletion of structural domains. However, a common core can be identified in the proposed structural models of these IRES elements from enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, and HAV. The common structural core identified within the picornavirus IRES is also conserved in the 5'NCR of the divergent viruses, HCV, and pestiviruses. Furthermore, the proposed structural motif shares a structural feature similar to that observed in the catalytic core of the group 1 intron. The conserved structural motif from these divergent sequences that looks like the common core region of group 1 introns is probably a crucial element involved in the IRES-dependent translation.

Animals↗

PubMed1990

cis-acting sequences required for in vivo amplification of genomic RNA3 are organized differently in related bromoviruses.

Cowpea chlorotic mottle virus (CCMV) is a positive-strand RNA virus that infects dicotyledonous plants. The genome comprises three capped RNAs: RNA1 (3.2 kb), RNA2 (2.9 kb), and RNA3 (2.1 kb). cis-Acting sequences required for amplification in vivo were explored for RNA3, which does not contribute trans-acting factors to viral RNA replication. Using a CCMV cDNA expression system, deletions throughout RNA3 were constructed and tested for successful replication in barley protoplasts coinoculated with RNAs 1 and 2. As previously found for RNA3 of the related brome mosaic virus (BMV) (R. French and P. Ahlquist, 1987, J. Virol. 61, 1457-1465), either of the two coding regions can be individually deleted without blocking RNA3 amplification. However, in striking contrast to BMV, the entire intercistronic noncoding region separating these genes is also dispensable for CCMV RNA3 amplification. Moreover, although simultaneous deletions of the 3a and coat protein genes were deleterious for BMV RNA3 accumulation, CCMV RNA3 derivatives bearing larger deletions encompassing the 3a gene, intercistronic region, and coat protein gene amplify to high levels. Thus, unlike BMV RNA3, cis-acting sequences required for CCMV RNA3 amplification map solely in the 5' and 3' noncoding regions. Normal levels of CCMV RNA3 accumulation require over 125 but no more than 220 bases from the 3' noncoding region, and no more than the first 89 bases of the 238-base-long 5' noncoding region.

Chromosome Deletion↗

PubMed2026

Identifying Co-Expressed lncRNAs Correlated With Traits of Interest in an Animal Model for Metabolic Diseases in Humans.

Nutrigenomics investigates how nutrients modulate gene expression. Among them, fatty acids (FA) play important roles in regulating gene transcription, while long non-coding RNAs (lncRNAs) may be associated with gene regulation and metabolic diseases. This study aimed to analyze the hepatic transcriptome of pigs, a species frequently used as a model for nutrigenomic studies, to identify novel lncRNAs and their potential target genes in response to diets containing different sources of FA. Seventy-two pigs were fed four diets supplemented with 1.5% soybean oil (control), 3% canola oil, 3% fish oil, and 3% soybean oil. RNA sequencing of liver samples was performed to identify novel lncRNAs. Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify modules associated with phenotypic traits related to lipid metabolism and inflammation. Functional enrichment analyses were then conducted to annotate genes within these modules using Gene Ontology (GO) terms and to assess overlap with Quantitative Trait Loci (QTL). The results revealed 106 novel lncRNAs potentially regulating genes associated with lipid metabolism and immune responses in pigs fed diets with different FA sources. These findings enhance understanding of the regulatory role of lncRNAs in pigs and reinforce their relevance as models for human metabolic diseases.

Animals↗

PubMed1991

Use of bromovirus RNA3 hybrids to study template specificity in viral RNA amplification.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.

Cloning, Molecular↗