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At least 955 records · Page 53Linked to original sources
PubMed1983

Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing.

Sequence comparison of the introns of two yeast genes (rp51A and rp51B) coding for the same ribosomal protein shows homology only in the last 50 bases of the intron. This region of the intron contains an internal conserved sequence (ICS) present near the 3' end of all sequenced yeast nuclear mRNA introns. Removal of a 29 bp sequence containing the ICS prevents splicing of an intron-containing hybrid gene. In cells containing the wild-type gene, we have detected RNA molecules that we suggest are normal splicing intermediates, generated by an endonucleolytic cut in the primary transcript at the ICS. The homology of the ICS with a sequence near the 5' end of U1 snRNA suggests a model in which an interaction in cis between the ICS and the 5' splice junction in yeast is the counterpart of the interaction in trans between U1 and 5' splice junctions in higher eucaryotes.

Base Sequence↗
PubMed1992

Effects of insulin and insulin-like growth factors on neurofilament mRNA and tubulin mRNA content in human neuroblastoma SH-SY5Y cells.

Insulin-like growth factors (IGFs) are implicated in the development of the vertebrate neural circuitry, and increase neurite growth in vitro and in vivo. The construction of the cytoskeleton is necessary for growth of axons and dendrites, and the neurofilament (NF) 68 kDa and 170 kDa proteins assemble to help form major fibrillar elements of the neurite cytoskeleton. We report that physiological concentrations of insulin, IGF-I or IGF-II increased the contents of 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs, relative to total RNA, in cultured human neuroblastoma SH-SY5Y cells. In contrast, the relative contents of histone 3.3 mRNA, and poly(A)+ RNA were not increased. Ligand concentrations which increased NF mRNAs were very similar to those which increased neurite outgrowth. Although each gene was evidently independently regulated, the 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs were nevertheless all transiently elevated over approximately the same time interval in response to insulin. These data, when considered together with studies by others with nerve growth factor, show that the 68 kDa and 170 kDa NF mRNAs are elevated in a biochemical pathway activated in common during neurite outgrowth directed by insulin, IGF-I, IGF-II, and nerve growth factor.

Gene Expression Regulation, Neoplastic↗
PubMed1996

Repeated ECS induces GluR1 mRNA but not NMDAR1A-G mRNA in the rat hippocampus.

The neural basis underlying the cognitive side effects of ECT is unknown. Recent studies suggest that the memory dysfunction may be caused by alterations in hippocampal synaptic efficacy [20]. In situ hybridisation was used to examine the possible receptor mechanisms responsible for this effect. Repeated ECS markedly increased mRNA expression for the GluR1 subunit of the AMPA receptor, but not the NMDAR1A-G subtypes of the NMDA receptor, relative to control treatments. This effect was present 24 h after the last seizure and may be responsible for the expression of the ECS-induced increase in synaptic efficacy.

Animals↗
PubMed1996

Comparison of RPTP zeta/beta, phosphacan, and trkB mRNA expression in the developing and adult rat nervous system and induction of RPTP zeta/beta and phosphacan mRNA following brain injury.

The receptor protein tyrosine phosphatase (RPTP) zeta/beta and a major isoform, phosphacan, a chondroitin sulfate proteoglycan that contains the RPTP zeta/beta extracellular domain but not the transmembrane and intracellular phosphatase domains, are expressed abundantly in the nervous system, primarily by astroglia. Because of similarities in the expression patterns of RPTP zeta/beta and the receptor tyrosine kinase TrkB, we investigated whether RNAs encoding these proteins were co-localized during development, which would suggest that these molecules might functionally interact in vivo. By in-situ hybridization, we noted extensive areas of overlap in the expression of trkB and RPTP zeta/beta mRNAs in the developing peripheral and central nervous systems. Analysis with a probe specific for the catalytic TrkB isoform suggested that RPTP zeta/beta and non-catalytic trkB mRNAs were co-expressed in particular regions of the nervous system while the catalytic trkB and RPTP zeta/beta transcripts were also, but to a lesser extent. RPTP zeta/beta and phosphacan expression were extremely similar, differing particularly in the level of expression in the ventricular and subventricular zones, hippocampus, and ependyma. Furthermore, both RPTP zeta/beta and phosphacan mRNAs were found in several subsets of neurons as well as astrocytes. Following CNS injury, we observed robust induction of RPTP zeta/beta mRNA in areas of axonal sprouting, and of both RPTP zeta/beta and phosphacan mRNAs in areas of glial scarring, implying that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps regulation of axonal regrowth in the nervous system.

Amino Acid Sequence↗
PubMed1990

Phage fl mRNA processing in Escherichia coli: search for the upstream products of endonuclease cleavage, requirement for the product of the altered mRNA stability (ams) locus.

In Escherichia coli infected with the filamentous phage f1, a number of the polycistronic phage mRNA species are generated through post-transcriptional processing by host nuclease activity. In this paper we review experimental evidence assessing whether known RNases are involved in mediating these processing events, and we use S1 nuclease mapping methods to visualize putative upstream products of endonuclease cleavage. By examining f1 processing in a phage-infected host bearing a temperature-sensitive allele of the altered message stability locus (ams), we show that production of the major processed species requires a component of the host cell which functions in the messenger RNA decay process.

Coliphages↗
PubMed1989

Prolactin (PRL) mRNA from human decidua differs from pituitary PRL mRNA but resembles the IM-9-P3 lymphoblast PRL transcript.

The expression of the human prolactin (hPRL) gene is normally restricted to the anterior pituitary and the decidualized endometrium of the uterus. The human B-lymphoblastoid cell line IM-9-P3 ectopically expresses a PRL mRNA which is about 150 nucleotides larger than its pituitary counterpart even though the mature protein products appear identical. In the present study we show that human decidual and IM-9-P3 PRL mRNAs are similar in size, both being elongated relative to the pituitary transcript. The size difference persisted after removal of the 3' end poly(A) tract, whereas the PRL precursors synthesized in a cell-free translation system using mRNAs from the three tissues showed identical apparent molecular weights upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The elongation could therefore not be attributed to differences in the protein coding region or in the degree of polyadenylation and is suggested to reside in the 5' untranslated region of the molecule.

Decidua↗
PubMed1992

Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain.

The cytokine interleukin 6 (IL6) has several effects on the central nervous system in addition to the well established regulation of the acute phase inflammatory response. Therefore, the distribution of IL6- and IL6 receptor mRNA in the rat brain has been investigated by in situ hybridization using [35S]-labeled oligonucleotides. The messages of both genes were found in the CA1-CA4 regions as well as in the dentate gyrus of the hippocampus, in the habenulae, the dorsomedial and the ventromedial hypothalamus, in the internal capsule, the optic tract and in the piriform cortex. These data indicate both neuronal and glial localization of IL6 and IL6 receptor and their involvement in an autocrine or paracrine action of the cytokine in centrally regulated functions including neuroendocrine control.

Animals↗
PubMed1993

Mechanisms of aluminum fluoride- and insulin-stimulated p33 mRNA accumulation in rat hepatoma cells: involvement of a G protein and kinase action and demonstration of effects on mRNA turnover.

The insulin signalling pathway to control nuclear p33 gene expression was examined. An AlF4-stimulated pertussis toxin-insensitive G protein was shown to be involved. The action of AlF4- was completely blocked by deferoxamine. Insulin action was markedly stimulated in the presence of AlF4-. cAMP and diacylglycerol concentrations were examined as possible regulators but no increases were detected. The effects of AlF4- and of insulin were completely inhibited by the general kinase inhibitor H-7. A second calcium calmodulin protein kinase inhibitor, W-7, had no detectable effect. Insulin and AlF4- were shown to stabilize p33 mRNA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2005

Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells.

Nonsense-mediated mRNA decay (NMD) generally involves nonsense codon recognition by translating ribosomes at a position approximately 25 nts upstream of a splicing-generated exon junction complex of proteins. As such, NMD provides a means to degrade abnormal mRNAs that encode potentially deleterious truncated proteins. Additionally, an estimated one-third of naturally occurring, alternatively spliced mRNAs is also targeted for NMD. Given the extraordinary frequency of alternative splicing together with data indicating that naturally occurring transcripts other than alternatively spliced mRNAs are likewise targeted for NMD, it is believed that mammalian cells routinely utilize NMD to achieve proper levels of gene expression.

Animals↗
PubMed1997

Protein environment of mRNA at the decoding site of 80S ribosomes from human placenta as revealed from affinity labeling with mRNA analogs--derivatives of oligoribonucleotides.

Affinity labeling of 80S ribosomes from human placenta has been studied using various mRNA analogs, namely, 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivatives of oligoribonucleotides (Up)(n-1)U[32P]pC (n = 3, 6 or 12) and AUGU3[32P]pC as well as ([4-(N-2-chloroethyl)-N-methylamino]benzylmethyl-[5'-32P]-phospham ide derivatives of pAUGUn (n = 3 or 6). Labeling of 80S ribosomes with the derivatives of oligouridylates was carried out in complexes obtained nonenzymatically in the presence of saturating amounts of Phe-tRNA(Phe). Complexes with derivatives bearing AUG codon were obtained using a fractionated lysate from rabbit reticulocytes which contained protein translation factors and was deprived from endogeneous ribosomes and mRNAs. In all cases, 40S subunits were labeled preferentially. Within the subunits, both 18S rRNA and proteins were found to be modified. Sites of cross-linking in 18S rRNA have been identified earlier. Here, it is shown that the main targets of cross-linking among the ribosomal proteins were S3 and S3a (with minor modification of S26) for the 3'-derivatives of (Up)5UpC and (Up)11UpC. For the same derivative of (Up)2UpC, the reverse modification pattern was observed. 5'-derivatives of pAUGUn were cross-linked to proteins S3 and S3a in comparable extent; 3'-derivative of AUGU3pC modified protein S3a preferentially.

Affinity Labels↗
PubMed2001

Expression of c-fos mRNA is increased and related to dynorphin mRNA expression following excitotoxic spinal cord injury in the rat.

Previous studies have demonstrated that excitotoxic spinal cord injury (SCI) created by the intraspinal injection of quisqualic acid (QUIS) is capable of inducing opioid peptide gene expression within the spinal cord and cortex. The opioids are classically involved in the suppression of pain transmission but specifically, dynorphin, has been implicated in the secondary pathophysiologic response to SCI. Activation of the immediate early gene, c-fos, has been implicated in the induction of preprodynorphin (PPD) gene expression and therefore, may be an important intermediate step in the generation of the opioid response to SCI. The purpose of this study was to investigate whether intraspinal QUIS injection induces c-fos expression within the spinal cord. Male, Long-Evans, adult rats (n=5) received an intraspinal injection of 1.2 microl of 125 mM QUIS directed at spinal segments T12-L2. Four hours post-injection brain and spinal cord tissues were removed and processed for in situ hybridization. Integrated density of c-fos and PPD mRNA expression was increased in the spinal dorsal horn following QUIS injection as compared to sham-injected animals. This indicates that SCI rapidly induces c-fos and PPD expression and suggests that c-fos plays a role in the induction of PPD expression.

Animals↗
PubMedDate not supplied

mRNA sequence for human cardiodilatin-atrial natriuretic factor precursor and regulation of precursor mRNA in rat atria.

The mammalian cardiac atrium has recently been shown to contain numerous peptides that exert marked effects on kidney function and vascular resistance. With one exception, the peptides have potent natriuretic and diuretic activities, and sequence similarities suggest that they may be derived from a common atrial natriuretic factor (ANF) precursor. The exception, cardiodilatin (CDD), differs from the ANF peptides in possessing potent vasorelaxant activity, but not natriuretic and diuretic activities. Here we report the cloning and sequence analysis of the cDNA for the human precursor protein (preproCDD-ANF) containing both the CDD and ANF sequences. The CDD sequence represents the N-terminal sequence preceded directly by a signal peptide, while the ANF sequence is present at the C-terminal end of the protein. Using hybridization analysis, we further show that the amount of rat preproCDD-ANF mRNA, which is synthesized selectively in the atria but not the ventricles, markedly decreases on water deprivation, suggesting that the water-electrolyte balance may be an important factor in the regulation of the expression of the preproCDD-ANF gene.

Amino Acid Sequence↗
PubMed2003

Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells.

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.

Base Sequence↗
PubMed1977

Hybridization properties of immunoglobulin mRNA: failure to detect covalently associated IgG mRNA transcripts of reiterated and unique mouse DNA.

Messenger RNAs for antibody heavy (gamma) and light (kappa) chains were isolated from the polysomes of an IgG-producing mouse myeloma cell line. Polysomes engaged in heavy or light chain synthesis were separated by immunoprecipitation using rabbit antibodies specific for the mouse IgG formed. The mRNAs obtained, more than 85% specifying IgG [Legler, M. & Cohen, E. P. (1976) Biochemistry 15, 4390-4399], were used as "probes" in hybridization experiments with sheared mouse liver DNA. To determine whether mRNAs for Ig heavy and light chains contained covalently bound transcripts of unique and reiterated DNA, hybrids were isolated with or without treatment with ribonuclease (RNase) prior to fractionation and the apparent rates of hybridization were compared. A monophasic C(o)t (DNA concentration x incubation time) curve with a C(0)t(1/2) of 4000 moles of nucleotide per liter x sec, corresponding to less than five hybridization sites per haploid genome, was obtained whether or not RNase was used in the isolation protocol. With a similar experimental design, the apparent hybridization rates of heterogeneous nuclear RNA from the same cell source were clearly different. The "stringency" of the reaction was reduced by incubating the hybridization mixtures at lower temperatures in a further attempt to detect a large class of repetitive sequences that would form hybrids with the IgG mRNA used, if such sequences were present. The results, however, were the same; i.e., the apparent rates of hybridization of mRNAs for mouse antibody gamma and kappa chains with sheared mouse liver DNA were essentially the same whether or not RNase was used in the isolation procedure. Reiteration of genes in mouse liver DNA for mouse IgG could not be detected.

Animals↗
PubMed1986

Human cytochrome P-450 4 mRNA and gene: part of a multigene family that contains Alu sequences in its mRNA.

Several overlapping lambda gt11 cDNA clones have been sequenced and shown to encode for the full-length human cytochrome P-450 4. The structure and location of the exons and flanking intron regions were also identified from a lambda EMBL-3 human genomic clone that encodes the full-length human P-450 4 gene. The human P-450 4 mRNA is flanked by 62 base pairs of 5'- and 1508 base pairs of 3'-noncoding sequence, with 1548 bases that encode a protein of 516 amino acids (Mr, 58,376). The predicted amino acid sequence of human P-450 4 is 69% and 70% homologous to its equivalent in mouse and rat, respectively, 75% homologous to rabbit P-450 4, and 68% homologous to human P1-450. The 7.6-kilobase gene encodes 3118 nucleotides of exon sequence that is separated by six introns into seven exons. Exon 7, which is 1802 nucleotides, contains three inverse/complement Alu sequences that are organized in tandem. Comparison of the genomic DNA sequence of the human P-450 4 gene with the human P1-450 and related genes in rat and mouse and the identification of the amino acid residues and triplet codon at each exon-intron junction show that the location of each intron in the human P-450 4 gene is conserved within this gene family. Although the length and homology of the introns within a related gene family may not be conserved, the location of intronic sequences may be an important determinant in the identification of related P-450 genes.

Amino Acid Sequence↗
PubMed1988

Localized maternal mRNA related to transforming growth factor beta mRNA is concentrated in a cytokeratin-enriched fraction from Xenopus oocytes.

The localized maternal RNA Vg1 resides in the cortical region of the vegetal pole of fully grown Xenopus oocytes and is inherited by only a subset of blastomeres in the early embryo [Weeks, D. L. & Melton, D. A. (1987) Cell 51, 861-867]. Because RNA-cytoskeletal interactions may play a role in RNA localization, we have examined the association of Vg1 RNA with components of the oocyte's cytoskeleton. Gel and immunoblot analysis of a detergent-insoluble fraction revealed a greatly simplified protein pattern composed largely of cytokeratins and vimentin. In sharp contrast to the nonlocalized histone H3 mRNA, Vg1 RNA was concentrated some 35- to 50-fold in this insoluble fraction. Extractions at higher salt concentrations yielded preparations further enriched in cytokeratins and in the Vg1 RNA. Upon ovulation, VG1 RNA is released into the soluble fraction. This change in Vg1 RNA distribution coincides with the observed breakdown of cortical cytokeratin filaments [Klymkowsky, M. W., Maynell, L. A. & Polson, A. G. (1987) Development 100, 543-557] and the loss of Vg1 RNA from the cortical region. Our findings are consistent with the hypothesis that RNA-cytoskeletal interactions are involved in the localization and segregation of information during development.

Animals↗
PubMed2002

Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control.

RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.

Amino Acid Sequence↗
PubMed2004

Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase.

MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5'-end of ACA sequences, yielding a 2',3'-cyclic phosphate at one side and a free 5'-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2'-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.

Base Sequence↗