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PubMed2004

A small bacterial RNA regulates a putative ABC transporter.

A small noncoding bacterial ribonucleic acid of 62-64 nucleotides, RydC, was identified in the genomes of Escherichia coli, Salmonella, and Shigella. In vivo, RydC binds to the RNA-binding protein Hfq, and it is unstable when Hfq is absent. Mobility assays reveal that complex formation between RydC and Hfq is specific, with an apparent binding constant of approximately 300 nm. Sequence alignments combined with structural probing demonstrate that RydC folds as a pseudoknot. Hfq binds the loops crossing the deep and shallow grooves of the pseudoknotted RNA and reorganizes its overall conformation. An interaction with a polycistronic mRNA, yejABEF, which encodes a putative ABC transporter, was detected by affinity purification of immobilized RNA-Hfq complexes. In vivo, the yejABEF operon is expressed on minimal medium. Remarkably, its expression is reduced when RydC is absent, and the operon is degraded when RydC expression is stimulated. This observation correlates with the growth defects associated with a stimulation of its expression in vivo, generating a thermosensitive phenotype that affects growth on minimal media supplemented with glycerol, maltose, or ribose. We conclude that RydC regulates the yejABEF-encoded ABC permease at the mRNA level. This small RNA may contribute to optimal adaptation of some Enterobacteria to environmental conditions.

ATP-Binding Cassette Transporters↗

PubMed1984

The complete sequence of the M RNA of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein.

The complete sequence of the viral M RNA of snowshoe hare (SSH) bunyavirus has been determined. The RNA is 4527 nucleotides long (mol wt: 1.5 X 10(6), base composition: 27.5% A, 33.5% U, 17.7% G, 21.3% C), and has 3' and 5' terminal sequences that, depending on how they are arranged, are complementary for some 44 residues. The viral RNA codes in its viral-complementary sequence, for a single primary gene product (the viral glycoprotein) that is comprised of 1441 amino acids (162,391 Da), and is rich in cysteine residues but poor in potential asparagine-linked glycosylation sites. Like the SSH S RNA, the M viral-complementary 5' noncoding region is shorter than the 3' noncoding sequence (61 as opposed to 142 nucleotides). The different functions of the M RNA are discussed in relation to those of the S RNA of SSH virus. No other large open reading frames have been identified in either the viral, or viral-complementary, M RNA sequences. Examination of the sequence of the M gene product reveals the presence of an 18 residue amino terminal hydrophobic sequence (putative signal) and a much longer 32 amino acid carboxy proximal hydrophobic region that is followed by a terminal sequence rich in charged amino acids (12 out of 20 residues). The size and constitution of the carboxy end regions are consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. In addition to these terminal hydrophobic sequences, a localized internal region of the gene product contains several hydrophobic sequences, 15 to 29 amino acids in length. Their possible role in the morphogenesis of bunyaviruses that occurs in the Golgi cisternae of infected cells is discussed.

Amino Acid Sequence↗

PubMed2002

Enox, a novel gene that maps 10 kb upstream of Xist and partially escapes X inactivation.

Dosage compensation in mammals is accomplished by the transcriptional silencing of a single X chromosome in female cells, a process termed X inactivation. A cytogenetically defined region of the X chromosome, the X-inactivation center (Xic), is necessary in cis for this process. Although the precise nature of the Xic remains unknown, a key component, the Xist gene, has been shown to be essential for X inactivation. In XX somatic cells, Xist RNA is specifically transcribed from the inactive X chromosome, which is otherwise essentially heterochromatic. Previous studies aimed at defining the proximal limit of the Xic have indicated that it lies within 30 kb upstream of the Xist promoter. Here we describe a novel gene, Enox (expressed neighbor of Xist), that maps to an unmethylated CpG island 10 kb upstream of Xist. Enox transcripts are antisense relative to Xist, highly heterogeneous, and apparently noncoding. In female somatic tissue Enox partially escapes from X inactivation. We discuss the implications of these findings in relation to our understanding of the Xic.

Animals↗

PubMed1984

Transcriptional activation of the translocated c-myc oncogene in mouse plasmacytomas: similar RNA levels in tumor and proliferating normal cells.

We examine the influence of the immunoglobulin locus on the expression of the translocated c-myc oncogene in mouse plasmacytomas. The level of c-myc RNA was 30- 35-fold greater in tumor cells than in normal, quiescent B cells. Mitogen stimulation of the lymphocytes with lipopolysaccharide induced a 15-fold increase in c-myc expression per cell to a level that was similar to that in the transcription of the translocated c-myc gene involved initiation from sequences in the first c-myc intron. Abundant RNA transcripts were also found from the noncoding strand of the c-myc intron in most tumor lines. S1 nuclease mapping was used to locate the intronic sequences that are used to initiate the tumor-specific c-myc RNAs. Six different initiation sites within the intron were mapped, none of which have the TATA sequence usually associated with eucaryotic RNA polymerase II promoters. The noncoding strand transcripts were also found to initiate in the c-myc intron. Transcription of the c-myc coding strand was independent of the position of the translocation breakpoint, even when the heavy chain switch and constant regions were deleted.

Animals↗

PubMed1985

Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA.

Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.

Acetyltransferases↗

PubMed1994

Influenza virus NS1 protein stimulates translation of the M1 protein.

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.

Base Sequence↗

PubMed1994

Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein.

The structure of human rhinovirus-14 3C protease (3Cpro) has been determined at 2.3 A resolution and refined to an R factor of 0.22. This cysteine protease folds into two topologically equivalent six-stranded beta barrels and in this sense is similar to trypsin-like serine proteases. However, there are differences in the lengths and positioning of individual beta strands as well as in loops connecting elements of secondary structure. The catalytic residues Cys-146, His-40, and Glu-71 are positioned as in serine proteases, but the oxyanion hole is moved 1-1.2 A away. Residues that bind to the 5' noncoding region of rhinovirus genomic RNA are located on the opposite side of the molecule from the active site. Interactions between individual 3Cpro molecules in the crystal lattice suggest a model for intermolecular proteolytic cleavage of the 3CD polyprotein.

3C Viral Proteases↗

PubMed1980

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).

Base Sequence↗

PubMed1998

Identification of specific nucleotide sequences within the conserved 3'-SL in the dengue type 2 virus genome required for replication.

The flavivirus genome is a positive-stranded approximately 11-kb RNA including 5' and 3' noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3' NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3'-SL), formed by the 3'-terminal approximately 100 nt. The nucleotide sequences within the 3'-SL are not well conserved among species. We examined the requirement for the 3'-SL in the context of dengue virus type 2 (DEN2) replication by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3'-terminal 93 nt constituting the wild-type (wt) DEN2 3'-SL sequence were replaced by the 96-nt sequence of the West Nile virus (WN) 3'-SL, was sublethal for virus replication. An analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3'-SL structures suggested that the wt DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3'-SL was required for viability. One 7-bp substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions of and/or substitutions in the wt DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved.

Animals↗

PubMed1994

Targeting of the site of nonhomologous genetic recombination in brome mosaic virus.

The genome of brome mosaic virus (BMV) consists of three positive strand RNA segments that share a high degree of sequence homology in the 3' noncoding region. The phenomenon of both homologous and nonhomologous intersegment RNA-RNA recombinant has been demonstrated within the 3' noncoding region of BMV RNAs. It has been postulated that nonhomologous crossovers occur at local heteroduplexes formed between the recombining BMV RNA substrates of the same polarity and that the formation of double-stranded regions facilitates strand switching by the replicase. To test the hypothesis of hybridization-mediated recombination in BMV, RNA-3 constructs carrying short antisense RNA1-derived sequences have been used to induce nonhomologous recombination events between RNA-1 and RNA-3 at or near the site of hybridization. We find that both the incidence of recombination and the location of recombinant junctions depends on the structure and the stability of heteroduplexes. Furthermore, our preliminary results demonstrate that mutations in the helicase-like domain of BMV protein 1a affect the location of recombinant junctions. This provides experimental evidence that BMV replicase protein 1a participates in recombination.

Bromovirus↗

PubMed2002

RNA folding in vivo.

RNA folding in vivo is influenced by the cellular environment, the vectorial nature of transcription and translation, trans-acting factors and ion homeostasis. Specific RNA-binding proteins promote RNA folding by stabilizing the native structure or by guiding folding. In contrast, RNA chaperones, which are believed to interact nonspecifically with RNA, were proposed to resolve misfolded RNA structures and to promote intermolecular RNA-RNA annealing. Small trans-acting noncoding RNAs are thought to modulate mRNA structures, thereby regulating gene expression. So far, there is some evidence that in vitro and invivo RNA folding pathways share basic features. However, it is unclear whether the rules deduced from in vitro folding experiments generally apply to invivo conditions.

3' Flanking Region↗

PubMed1989

3' noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element.

The stabilization of phosphoenolpyruvate carboxykinase mRNA by glucocorticoids appears to result from the interaction of an induced factor with an RNA element located in the 3' noncoding sequence of the mRNA. This element can confer glucocorticoid-dependent stabilization upon a heterologous mRNA, and thus strategies developed to investigate the control of mRNA transcription can now be applied to the analysis of hormone-regulated mRNA stabilization.

Animals↗

PubMed1998

Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

Animals↗

PubMedDate not supplied

The role of mRNA 5'-noncoding and 3'-end sequences on 40S ribosomal subunit recruitment, and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis.

Since the elaboration of the scanning model to explain eukaryotic translation initiation, alternative hypotheses have gained support. Cap and 5' end-independent recruitment of the 40S ribosomal subunit conferred by the presence of an internal ribosome entry segment (IRES) in the 5'UTR of the mRNA is widely accepted, and has been formally and definitively proven for a picornavirus. However, the mechanism of IRES function remains essentially a black box. Using the complex viral IRESes as model systems, approaches taken to shed light on the mystery include systematic comparisons and molecular genetic analyses. The hypothesis that actively translated mRNAs are circular, rather than linear, molecules is based on rather indirect evidence. This model has invoked a revision of the image of 40S ribosomal subunit recruitment, to include recycling from the mRNA 3'- to the 5'-end in addition to true de novo 5'-end directed entry. Biochemical and genetic studies are used to define the network of interactions necessary for efficient ribosome recruitment. This has lent weight to the concept of mRNA 5'-3' cross-talk and clarified the mechanics of how this enhances translation efficiency. These refinements and revisions to the model of translation initiation form the core of this review, with current knowledge being considered from the perspective on how host-cell translation could yield to selective viral translation via the phenomenon of translational shut-off.

5' Untranslated Regions↗

PubMed1992

An antisense oligonucleotide complementary to a sequence in I gamma 2b increases gamma 2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion.

An antisense phosphorothioate (S)-oligonucleotide to a sequence in the intervening (I) region of the gamma 2b immunoglobulin (Ig) heavy chain gene inhibits Ig secretion by B cells stimulated with lipopolysaccharide (LPS) or LPS plus interleukin 4. It is also a striking stimulant of DNA synthesis by resting B cells. The antisense S-oligonucleotide causes a 10-20-fold increase in the expression of the gamma 2b germline transcript. Among mutants of the antisense S-oligonucleotide, some show all the effects whereas others are inactive. A similar hierarchy exists in the quantitative biological activities of mutant S-oligonucleotides and in their capacity to hybridize to the sense oligonucleotide, strongly suggesting that an I gamma 2b sequence in the RNA transcript or in the noncoding strand of the DNA is the target of the antisense S-oligonucleotide. The possible relationship of the overexpression of the germline gamma 2b transcript to the biological functions of the I gamma 2b antisense S-oligonucleotide is discussed.

Animals↗

PubMed1995

An RNA stem-loop structure involved in the packaging of bovine leukemia virus genomic RNA in vivo.

An RNA secondary structure of the bovine leukemia virus (BLV) 5'-terminal RNA sequence was constructed by computer-assisted RNA secondary structure analysis. Mutations were created in the noncoding region (NCR) of BLV, which contains a conserved consensus sequence, to disrupt predicted secondary structure of this region. After transfection of these constructs into FLK-BLV cells and analysis of viral particles a reduction in mutant RNA content was observed relative to that of unmutated vector RNA. The packaging efficiency of the mutant with a substitution in the consensus sequence was reduced threefold and that of the mutant with a deleted 5' NCR was reduced fivefold. We conclude that predicted RNA secondary structure and/or nucleotide sequence of the BLV noncoding region is essential for BLV RNA packaging in vivo.

Animals↗

PubMed1998

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.

Amino Acid Sequence↗

PubMed1984

RNA splicing as an error-screening mechanism.

Eukaryote nuclear genes are generally split into coding (exon) and noncoding (intron) regions. During the formation of messenger RNA the introns are precisely excised and the exons religated. A widely accepted explanation for the split structure of eukaryotic genes is that proposed by Gilbert who hypothesized that the division of coding information into small units speeded up the rate of protein evolution by allowing for the recombination of the independent peptide domains encoded by these units ("exon shuffling"). However it has recently become clear that the exon:intron structure of genes most likely preceded the uninterrupted form. This makes it difficult to accept Gilbert's argument as it applies to the origin(s) of split genes since early genes were very inaccurately copied and a highly error-prone system needs less variation not more. Here I propose that split genes and the concomitant process of RNA splicing arose as a mechanism for maintaining the stability of the genetic information in the face of a high level of noise in the gene copier mechanism.

Base Sequence↗