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At least 271 records · Page 15Linked to original sources
PubMed2000

CD8 coreceptor extinction in signaled CD4(+)CD8(+) thymocytes: coordinate roles for both transcriptional and posttranscriptional regulatory mechanisms in developing thymocytes.

T-cell development in the thymus is characterized by changing expression patterns of CD4 and CD8 coreceptor molecules and by changes in CD4 and CD8 gene transcription. In response to T-cell receptor (TCR) signals, thymocytes progress through developmental transitions, such as conversion of CD4(+)CD8(+) (double-positive [DP]) thymocytes into intermediate CD4(+)CD8(-) thymocytes, that appear to require more-rapid changes in coreceptor expression than can be accomplished by transcriptional regulation alone. Consequently, we considered the possibility that TCR stimulation of DP thymocytes not only affects coreceptor gene transcription but also affects coreceptor RNA stability. Indeed, we found that TCR signals in DP thymocytes rapidly destabilized preexisting CD4 and CD8 coreceptor RNAs, resulting in their rapid elimination. Destabilization of coreceptor RNA was shown for CD8alpha to be dependent on target sequences in the noncoding region of the RNA. TCR signals also differentially affected coreceptor gene transcription in DP thymocytes, terminating CD8alpha gene transcription but only transiently reducing CD4 gene transcription. Thus, posttranscriptional and transcriptional regulatory mechanisms act coordinately in signaled DP thymocytes to promote the rapid conversion of these cells into intermediate CD4(+)CD8(-) thymocytes. We suggest that destabilization of preexisting coreceptor RNAs is a mechanism by which coreceptor expression in developing thymocytes is rapidly altered at critical points in the differentiation of these cells.

Animals↗
PubMed1997

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

Animals↗
PubMed2002

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

5' Untranslated Regions↗
PubMed1982

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus.

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.

Amino Acid Sequence↗
PubMed1993

Effects of 5-fluorouracil on mRNA.

Currently, there are a number of studies which suggest that FU can have pronounced effects on mRNA and its metabolism. However, the relevance of these changes to the antitumor effect of FU are still not clear. Generally, the mRNAs which have been studied to date involve those genes which are associated with the TS-directed effects of FU and have generally been limited to the changes in mRNA levels. The recent development of PCR methodology to investigate changes in pre-mRNA and splicing provides the tool to study a number of RNA effects of FU simultaneously. The major question is which mRNAs are important for study. DHFR mRNA has a half life of 11.5 in KB1BT cells (Will and Dolnick, 1989) and is thus, on a kinetic basis alone, unlikely to provide a significant RNA target for RNA-directed effects of FU. There is a greater likelihood that shorter lived mRNAs which not only turnover rapidly, but are important to cell proliferation will eventually be shown to be key targets for the effects of FU at the RNA level. Interestingly, many of the growth factors are encoded by short-lived and tightly regulated mRNAs (e.g. GM-CSF, Shaw and Kamen, 1986). In fact the half-lives of some of these mRNAs are regulated by U-rich sequences in their 3'-noncoding regions. The presence of U-rich sequences in these growth factor mRNAs and the small nuclear RNAs suggests these are worthwhile targets for studies, which could now be performed on clinical samples. Laboratory data which shows alterations in the small nuclear RNAs, under conditions which only provide for very low-level substitution of U residues by FU also suggest that RNA effects of FU may be a much more tightly related to cytotoxicity in vivo than previously thought.

Animals↗
PubMed2002

RNA recombination in brome mosaic virus: effects of strand-specific stem-loop inserts.

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.

Base Sequence↗
PubMed1985

Developmental and tissue-specific expression of a family of transcripts related to rat insulin-like growth factor II mRNA.

We have constructed a cDNA library from the mRNA of a rat liver cell line (BRL-3A) and characterized cDNA clones encoding the protein precursor of the rat insulin-like growth factor II (pre-pro-rIGF-II). This precursor, inferred from the nucleotide sequence, consists of a signal peptide, the rIGF-II sequence, and a trailer polypeptide of unknown significance. The characterized cDNA sequence (1016 nt) is part of a 3.4 kb mRNA species. Northern analysis reveals that a probe containing the extreme 5' noncoding region hybridizes to a second RNA (1.6 kb), while a probe corresponding to the 5' noncoding region proximal to the coding region hybridizes to two other RNA species (1.75 and 1.1 kb). All four RNAs are differentially expressed in all of the neonatal tissues that were examined, while the 3.4 kb pre-pro-rIGF-II mRNA and the 1.1 kb transcript are absent from adult tissues.

Amino Acid Sequence↗
PubMed1997

Translation and replication properties of the human rhinovirus genome in vivo and in vitro.

The poor translation efficiency of genome-length human rhinovirus RNA in vitro using HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficient in vitro translation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particles in vitro. A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesis in vivo. In order to test this hypothesis and extend in vitro translation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed the in vitro synthesis of T7 transcripts containing the intact poliovirus type 1 (PV1) 5' noncoding region (5' NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14) polyprotein-coding region and 3'-terminal sequences. These chimeric RNAs translated efficiently in vitro and were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimera in vitro. The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirus in vivo are determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5' NCR may be a limitation in rabbit reticulocyte lysate-based in vitro translation extracts, it does not appear to be a major limiting determinant for growth of rhinovirus in vivo or replication in the HeLa cell-free extract.

Animals↗
PubMed1989

Antisense RNA complementary to 3' coding and noncoding sequences of creatine kinase is a potent inhibitor of translation in vivo.

Antisense RNA is a potentially powerful tool for creating dominant negative mutations, but one of the limitations of this strategy has been the relative inefficiency of antisense transcripts in blocking target gene expression. To identify more effective target sequences, helper-free retrovirus-mediated gene transfer was used to introduce antisense RNAs complementary to multiple functional regions of the human creatine kinase B (CK-B) mRNA into U937 cells. Antisense RNA complementary to the last third of the coding and all of the noncoding regio of this mRNA is highly effective; one or two antisense transcripts is sufficient to block the expression of one CK-B mRNA. In contrast, antisense RNA from which sequences complementary to the last 17 codons and all the 3' noncoding region have been deleted has no effect on CK-B expression. Neither antisense RNA alters the abundance of the target message, processing of the primary transcript, egress of the CK-B message from the nucleus, or the polysome profile of CK-B mRNA in sucrose gradients. These results point to a direct effect of the antisense transcript on translation and suggest that this effect may be explained at least in part by an inhibition of elongation or termination as a consequence of the duplex formed in the distal coding and/or 3' noncoding region.

Blotting, Northern↗
PubMed1994

A novel synapse-associated noncoding RNA.

Synaptic nuclei of innervated muscle transcribe acetylcholine receptor (AChR) genes at a much higher level than extrasynaptic nuclei. To isolate candidate synaptic regulatory molecules responsible for the unique transcriptional potential of synaptic nuclei, we have taken a subtractive hybridization approach. Here, we report the cloning and characterization of a novel synapse-associated RNA, 7H4. 7H4 is expressed selectively in the endplate zone of skeletal muscle and is upregulated during early postnatal development and after denervation. Interestingly, the 7H4 gene has no introns, and yet two different-size RNAs with identical polyadenylated 3' ends are generated. Most intriguingly, the nucleotide sequence does not contain any significant open reading frames, suggesting that 7H4 may function as a noncoding RNA.

Animals↗
PubMed1996

Analysis of cis-acting elements required for replication of barley stripe mosaic virus RNAs.

The replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNA alpha and -gamma transcripts. The 5' and 3' noncoding regions were required for replication, and lack of a 5' cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNA alpha abrogated replication in trans. A 2-base change that produced a truncated alpha a protein lacking the first 16 amino acids also compromised the ability of RNA alpha to be replicated. In contrast, RNA beta transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs alpha and gamma, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between beta a and beta b was not absolutely required for replication, but small deletions within this region reduced the abundance of RNA beta by at least 10-fold. Deletions within the first 507 nt of the gamma a ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the gamma a ORF, the gamma a--gamma b intergenic region, and the gamma b ORF could be amplified in trans. Two mutants containing extensive deletions encompassing the central region of the gamma a ORF and most of gamma b behaved like defective interfering RNAs because they multiplied to high levels in trans and caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs alpha and gamma.

Base Sequence↗
PubMed2001

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.

Cytoplasmic Vesicles↗
PubMed1991

Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo.

DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the 5'-proximal P3 gene, encoding the putative viral transport function, did not affect replication whereas deletions in the 3'-proximal coat protein gene reduced replication of RNA 3 by about 100-fold. Sequences required for the synthesis in protoplasts of RNA 4, the coat protein messenger, were more extensive than the subgenomic promoter characterized previously in an in vitro replicase assay. At the 5'-end of RNA 3 a sequence of 169 nucleotides was sufficient for replication whereas a sequence of 112 nucleotides was not. 3'-Terminal deletions up to 133 nucleotides reduced replication to a low but significant level. Further 3'-deletions abolished replication.

Base Sequence↗
PubMed1999

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA.

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

3' Untranslated Regions↗
PubMed1996

G alpha i RNA antisense expression demonstrates the exclusive coupling of peptide YY receptors to G(i)2 proteins in renal proximal tubule cells.

A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M. (1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense G alpha i protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5'-noncoding region of G alpha i2 or G alpha i3 used as specific antisense templates. A Cl.10/alpha i2-clone was selected which displayed a drastic decrease (> 90%) of the expression of G alpha i2 without changes of G alpha i3, G alpha s, and G beta subunits (G alpha i1 is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of 125I-PYY binding by guanosine 5'-O-(thiotriphosphate) (GTP gamma S); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (> 80%) of G alpha i3 as compared to untransfected cells. The sensitivity to GTP gamma S and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to G alpha i2.

Animals↗
PubMed1980

Topography of the three late mRNA's of polyoma virus which encode the virion proteins.

The three cytoplasmic polyadenylated mRNA's which separately encode the three capsid proteins (VP1, VP2, and VP3) of polyoma virus were mapped on the viral genome by one- and two-dimensional gel electrophoreses of nuclease S1-resistant RNA-DNA hybrids. The mRNA's, which we designated mVP1, mVP2, and mVP3 to indicate the coding functions deduced from the cosedimentation of the RNAs and the messenger activities, comprise an overlapping set of 3'-coterminal molecules which also share a heterogeneous family of noncoding 5'-terminal regions (Flavell et al., Cell 16:357--371, 1979; Legon et al., Cell 16:373--388, 1979). The three species differ in the length of the 3' colinear coding region which is spliced to the 5' leader sequences. The common polyadenylated 3' end maps at map unit 25.3. The 5' ends of the colinear bodies of mVP1, mVP3, and mVP2 map at 48.5, 59.5, and 66.5 map units, respectively. An examination of the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606--618, 1980) in the vicinities of splicing sites approximated by the S1 gel mapping data for sequences common to the ends of known intervening sequences allowed prediction of the precise splice points in polyoma virus late mRNA's. In all three cases, the leader sequences are joined to the mRNA bodies at least 48 nucleotides before the translational initiation codon used in each particular messenger. The start signal which functions in each mRNA is the first AUG (or GUG) triplet after the splice junction.

Capsid↗
PubMed1991

Oral poliovirus vaccine in the United States: molecular characterization of Sabin type 3 after replication in the gut of vaccinees.

Derivatives of Sabin 3 shed from recipients of oral poliovirus vaccine in the United States (U.S.) were examined for genetic changes identified in strains excreted by vaccinees in the United Kingdom [U.K.; Evans et al., 1985; Cammack et al., 1988, Macadam et al., 1989]. Among the eight primary vaccinees studied, the duration of excretion and molecular evolution of type 3 strains varied greatly. The period of virus excretion after vaccination ranged from as few as 2 days to as many as 36 days. Nucleotide sequence analysis of viral RNAs extracted from shed virus indicated that only fifty percent of the vaccinees exclusively excreted strains in which the attenuating mutation at nucleotide 472 in the 5' noncoding region of the genome had reverted from uracil (U) to cytosine (C), the nucleotide found in neurovirulent strains. Compared to the wild-type Leon strain, the low activity of stool isolate KW4 in a complete monkey neurovirulence test demonstrated that presence of C at 472 does not render a type 3 strain pathogenic. Conversely, an isolate was identified which efficiently replicated in monkey nervous tissue and maintained the attenuated U at 472. Oligonucleotide fingerprinting and sequence analysis of viral RNAs from stool isolates indicated that one vaccinee (KW) eventually excreted intertypic recombinant strains consistent with those reported in the U.K. studies. Unique to this study, one vaccinee (KS) excreted nonrecombinant virus possessing U at 472 for up to 21 days. The significance of the KS strain profile in relation to differences in the U.S. vaccine compared to the vaccine distributed in the U.K. and other countries is discussed.

Animals↗