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PubMedDate not supplied

[Structure of nuclear pre-mRNA. X. New type double-helical structures in the pre-mRNA].

High molecular weight nuclear pre-messenger RNA (pre-mRNA or RNA) isolated from Ehrlich ascites carcinoma cells contains besides moderately long (100--200 base base pairs) snap-back double-stranded structures, also longer double-stranded structure containing at least 300-800 base pairs. Very long double-stranded sequences are not able to snap-back after RNA melting. While the moderately long double-stranded RNS (dsRNA) is renatured at Cot 1/2 approximately or equal to 5 X 10(-4), the very long dsRNA shows a higher complexity (Cot 1/2 approximately or equal to 2 X 10(-2). They also hybridize to a less reiterated class of DNA than moderately long dsRNA. Two classes of dsRNA are represented by different sequences as followed from cross-renaturation experiments. Very long dsRNA forms stable hybrids with 20% of total poly(A)+mRNA of cytoplasm. The properties of different classes of ds structures present in nuclear pre-mRNA are compared and their possible nature is discussed. The presence of very long dsRNA may reflect either the symmetric transcription of structural genes, or the transcription from thos DNA sequences which are complementary to each other but located in different parts of the genome.

Animals↗
PubMed1978

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. II. Characterization of pre-mRNA isolated by phenol extraction and poly(U)-Sepharose chromatography.

Precursor mRNA (pre-mRNA) was extracted from erythroid enriched bone marrow cells of the rabbit by the methods of Georgiev and Mantieva modified by Markov and Arion and of Holmes and Bonner, respectively. Density gradient centrifugation, base analysis and the effects of alpha-amanitin and actinomycin D on the synthesis of the cellular RNA showed signs of degradation in the rRNA-free 85 degrees C-fraction of the preparation according to Georgiev and Mantieva and a substantial rRNA contamination of the 65 degrees C-fraction. This RNA-fraction as well as the total RNA-preparation extracted according to Holmes and Bonner was purified from rRNA by affinity chromatography on poly(U)-Sepharose. Poly(A)+-RNA of all size-classes, among it a substantial amount of high molecular weight RNA (greater than 45 S), was isolated by this purification procedure. Especially the extraction according to Holmes and Bonner yields high molecular weight material but the critical step of this procedure often resulting in degradation of the RNA is the DNase treatment of the heavily DNA-contaminated total RNA-preparation either due to RNase contamination of the DNase or to the existence of RNase in the less intensive deproteinized RNA. The investigated cellular system is characterized by a very intensive rRNA synthesis which is typical for cells in the early stages of hematopoiesis. In contrast to investigations with purified RNA-polymerases and subcellular systems, but in accordance with data of in vivo experiments, alpha-amanitin inhibits both the pre-mRNA and the pre-rRNA synthesis.

Animals↗
PubMed1993

Identification of an mRNA species which encodes a voltage-operated Ca2+ channel in rat liver mRNA.

cDNA which encodes part of the alpha 1-subunit of the rabbit skeletal muscle L-type voltage-operated Ca2+ channel (VOCC cDNA) was employed to search for the presence in whole liver and hepatocytes of poly (A+) RNA homologous to mRNA which encodes VOCCs. Such homologous mRNA would be a candidate for mRNA which encodes the putative hepatocyte receptor-activated Ca2+ inflow system (RACIS). Northern blot analysis showed that poly (A+) RNA prepared from intact liver tissue, but not hepatocytes, contained a poly (A+) RNA species comparable in size to that which encodes the alpha 1-subunit of the L-type VOCC. It is concluded (a) that hepatocytes do not possess VOCCs or that the levels of VOCC poly(A+) RNA in hepatocytes are too low to be detected by Northern analysis and (b) that another cell type present in liver tissue does possess a VOCC. In a low stringency screen of a rat liver cDNA library employing VOCC cDNA as a probe, seven positive cDNA clones were obtained. While regions of the 2.3 kb cDNA insert from one of these clones showed sequence similarities with regions of VOCC cDNA, the 2.3 kb sequence did not appear to encode a Ca2+ channel. The present results suggest that the approach of low stringency cDNA library screening is unlikely to allow isolation of RACIS cDNA.

Animals↗
PubMed1995

Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA.

Cell culture studies demonstrating that the serine protease thrombin can induce neuronal and glial process retraction, glial proliferation, and changes in gene expression suggest a role for thrombin in CNS development, plasticity, and response to injury. Most cellular responses to thrombin are mediated by proteolytic activation of the cloned thrombin receptor (TR), a member of the seven transmembrane domain, G-protein-coupled receptor superfamily. As a step toward understanding the role of thrombin and its receptor in the CNS, Northern blot, in situ hybridization, and immunohistochemical techniques were used to analyze the cellular localization of TR mRNA in weanling-age rat brain. TR mRNA was broadly distributed across the neuraxis, although expression was very focal and often anatomically limited within specific neural structures. The greatest hybridization was associated with individual neurons in neocortex, cingulate/retrosplenial cortex, and subiculum, subsets of nuclei in hypothalamus, thalamus, pretectum, and ventral mesencephalon, and discrete cell layers in the hippocampus, cerebellum, and olfactory bulb. Patterns of hybridization included neuronal, glial, and ependymal cells, although white matter was uniformly negative, as were most cerebrovascular endothelial cells. Expression of TR mRNA by astroglia and dopaminergic neurons was confirmed by colocalization with immunoreactivity for glial fibrillary acidic protein (GFAP) in hippocampus and tyrosine hydroxylase in the substantia nigra. Comparison between TR and prothrombin (thrombin's precursor) cRNA hybridization demonstrated distinct but overlapping brain distributions of these transcripts, most clearly evident in postnatally developing, laminated structures. These results suggest widespread utilization of, and multiple physiologic, and possibly pathophysiologic, functions for, the thrombin/TR cell signaling system in the CNS.

Animals↗
PubMed1995

Comparison of the ankyrin (AC)n microsatellites in genomic DNA and mRNA reveals absence of one ankyrin mRNA allele in 20% of patients with hereditary spherocytosis.

Combined deficiency of ankyrin and spectrin represents the most common biochemical abnormality in hereditary spherocytosis (HS). To examine whether a decrease in ankyrin mRNA represents a frequent cause of this type of HS, we took advantage of the reported (AC)n microsatellite polymorphism in the 3' untranslated region of ankyrin cDNA. We first measured the number of AC repeats in genomic DNA encoding erythrocyte ankyrin in 36 unrelated Czech HS patients with combined ankyrin and spectrin deficiency and found 21 of these subjects (58%) to be heterozygotes for the (AC)n microsatellite size. Further analysis of reticulocyte RNA showed that ankyrin cDNA from 7 of these 21 heterozygotes (33%) contained only one of the two ankyrin alleles. We conclude that approximately 1/3 of ankyrin-deficient autosomal dominant HS is caused by reduced expression of one ankyrin allele which, in turn, is caused by either a reduced transcription of one allele of the mutated ankyrin gene or abnormal processing or decreased stability of the mutant ankyrin mRNA. Because ankyrin deficiency is detected in approximately 60% of HS subjects, this result suggests that approximately 20% of all HS is caused by a decreased expression of one ankyrin mRNA allele.

Alleles↗
PubMed1997

Polypeptide pattern of mRNA isolated from polysomal and non-polysomal mRNA-protein(mRNP) fractions in rat myoblast.

Polypeptides synthesized from polysome-bound and unbound fractions of polyA mRNA were studied by in-vivo cells labelling and cDNA-mRNA hybrid-arrest translation. The polypeptides synthesized from unbound(free) fraction appeared to be significantly reduced in the absence of cycloheximide in L6 myoblast. The cDNA excess-mRNA hybrid-arrest translation exhibited rare polypeptide sequences in the unbound fractions.

Animals↗
PubMed2025

Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner.

RNA, Messenger↗
PubMed2026

Genome-Wide Impact of Human DBR1 Depletion on RNA Processing Networks Reveal a Connection Between Pre-mRNA Splicing, mRNA Surveillance and Stress Granule Dynamics.

The RNA lariat debranching enzyme DBR1 is essential for intron turnover and RNA metabolism, yet its broader impact on transcriptome regulation remains incompletely defined. To elucidate the consequences of DBR1 depletion, we performed transcriptome-wide RNA sequencing of DBR1-knockdown and wild-type HEK293 cells. Differential expression analysis revealed widespread perturbations in pathways linked to RNA splicing, mRNA surveillance, translational control, and stress-granule biology. Many of the most significantly altered transcripts encode splicing factors and RNA quality-control components, underscoring DBR1's influence on post-transcriptional regulation. Alternative splicing analysis showed changes across multiple event types, with exon skipping accounting for >50% of events, followed by mutually exclusive exons, alternative 5' and 3' splice sites, and retained introns, indicating that DBR1 depletion induces pervasive splicing defects. Direct spliceosome inhibition using isoginkgetin (blocks tri-snRNP recruitment) and pladienolide B (targets SF3B1) reproduced the DBR1-KD mis-splicing patterns of cell signaling genes and factors involved in RNA metabolism, supporting a functional link between DBR1 activity and alternative splicing. Notably, DBR1 knockdown revealed a subset of transcripts that are both NMD-sensitive and enriched within stress granules. Consistent with this observation, G3BP1 immunopurification and confocal microscopy further support a role for DBR1 and UPF1 in stress-granule dynamics, suggesting that these factors may participate at distinct stages to influence mRNA fate under stress conditions. Together, these findings indicate that DBR1 functions beyond lariat RNA turnover as a common regulator of RNA processing, transcriptome stability, and stress granule homeostasis, revealing intricate crosstalk between RNA splicing and RNA quality control pathways in human cells.

Humans↗
PubMed2005

Loss of E-cadherin mRNA and gain of osteopontin mRNA are useful markers for detecting early recurrence of HCV-related hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: We investigated whether expression of E-cadherin (E-cad) and osteopontin (OPN) mRNAs could predict the early recurrence of HCV-associated hepatocellular carcinoma (HCV-HCC) after curative surgery. METHODS: Forty-four HCV-HCC patients who underwent curative hepatectomy were divided into three categories: category 1--recurrence (3 mR+: n = 6) or non-recurrence (3 mR-: n = 38) within 3 months; category 2--recurrence (6 mR+: n = 4) or non-recurrence (6 mR-: n = 34) between 3-6 months; category 3--recurrence (1 yR+: n = 4) or non-recurrence (1 yR-: n = 30) between 6-12 months. Levels of expression of E-cad and OPN mRNAs were analyzed quantitatively by real-time PCR and calculated using the formula: t = (copy of E-cad or OPN/copy of GAPDH) x 1,000. RESULTS: The t-value for E-cad was significantly lower in 3 mR+ than in 3 mR- (0.4 and 28.1, P < 0.05), in 6 mR+ than in 6 mR- (4.0 and 30.6, P < 0.05), and in 1 yR+ than in 1 yR- (9.2 and 32.9, respectively, P < 0.05). The t-value for OPN was higher in 3 mR+ than in 3 mR- (92.6 and 65.7, P = 0.57), in 6 mR+ than in 6 mR- (457.1 and 58.15, respectively, P = 0.24), and in 1 yR+ than in 1 yR- (221.8 and 53.5, respectively, P = 0.06). CONCLUSIONS: Low expression of E-cad mRNA and high expression of OPN mRNA are associated with a higher likelihood of early recurrence of HCV-HCC.

Aged↗
PubMed2006

Low mRNA levels of RGS4 splice variants in Alzheimer's disease: association between a rare haplotype and decreased mRNA expression.

Regulator of G-protein signaling 4 (RGS4) showed decreased mRNA levels in Alzheimer's disease in a large collection of human brain autopsies from prefrontal cortex. The expression levels of three RGS4 splice variants were examined in the same samples, and the association between RGS4 gene expression and/or the disease with single nucleotide polymorphisms located in this gene was explored. We show that all splice variants are down-regulated in patients. We also demonstrate that one rare haplotype (ATAG) is associated with decreased mRNA levels in both cases and controls. Our results suggest that an altered regulation in transcription initiation may be an important mechanism for low RGS4 protein levels in Alzeimer's disease.

Alleles↗
PubMed2000

Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction.

An enzyme-ribosome-mRNA complex was specifically purified by binding to the immobilized enzyme substrate and the cDNA was cloned in a single-tube reaction by one-step reverse transcription-PCR. The ganglioside GM3, used by sialyltransferase II (ST-II) as a substrate, was coated on a 96-well microtiter plate and ST-II was in vitro transcribed and translated from a cDNA library. The isolation of an enzyme-specific protein-ribosome (PRIME) complex was achieved with as little as 0.1 ng ST-II-specific cDNA in 5 microg of a total plasmid preparation or with the cDNA prepared from sublibraries previously inoculated at a density of 2000 clones/culture well. The affinity purification of the PRIME complex was highly specific for GM3 and did not result in cDNA amplification when a different ganglioside (GM1) was used for coating of the microtiter plate. The amplified cDNA was used for cloning or a second round of ribosome display, providing a fast analysis of enzyme affinity to multiple substrates. PRIME display can be used for host-free cDNA cloning from mRNA or cDNA libraries and for binding site mapping of the in vitro translated protein. The use of a single-tube reaction in ligand-coated microtiter plates indicates the versatility of PRIME display for cDNA cloning by automated procedures.

Chromatography, Affinity↗
PubMed1993

Autoinduction of platelet derived growth factor (PDGF) A-chain mRNA expression in a human malignant melanoma cell line and growth inhibitory effects of PDGF-A-chain mRNA-specific antisense molecules.

Autoinduction of PDGF-A-chain mRNA's was demonstrated in cells of a human malignant melanoma cell line (HTZ 19), indicating a PDGF-induced amplification of an autocrine growth factor loop mechanism. PDGF-alpha receptors were detected by immunocytochemistry in HTZ 19 melanoma cells. Exogenously administered PDGF-AA exerted an increase in cell proliferation by 70% at 200 ng/ml PDGF in serum-supplemented cell culture medium. Antisense phosphorothioate-oligodeoxynucleotides (S-ODN's), specifically targeted against PDGF-A-chain mRNA, reduced cell proliferation of HTZ 19 melanoma cells by 62% at a concentration of 5.0 microM, also only in serum-enriched medium. These findings strongly suggest that PDGF-AA homodimers may be an autocrine growth factor for HTZ 19 melanoma cells but require synergistic interaction with serum components.

Base Sequence↗
PubMed1994

Stabilization of interleukin-2 receptor alpha chain mRNA by HTLV-1 Rex in mouse L cells: lower amounts of Rex do not stabilize the mRNA.

T cell growth factor receptor, interleukin-2 receptor alpha chain (IL-2R alpha) is constitutively expressed on human T-cell leukemia virus type-1 (HTLV-1) infected T cells. We have established L cell lines which express both IL-2R alpha and the Rex protein of HTLV-1. We found that IL-1R alpha mRNA is stabilized in a cell line, Ltk/1-2a, which expresses a high amount of the Rex protein. In the presence of lower amounts of Rex, stabilization of the mRNA was not observed. These results may well explain the mechanism by which most of the lymphocytes infected with HTLV-1 escape from malignant transformation.

Animals↗
PubMed1997

Polymorphic expression of multidrug resistance mRNA in lung parenchyma of nonpregnant and pregnant rats: a comparison to cystic fibrosis mRNA expression.

Multidrug resistance (MDR1b) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins are members of the "ATP-binding cassette" superfamily of transporters. They are associated with chloride channel activities and ATP secretion and have complementary patterns of expression in several organs. In the rat uterus, CFTR expression is replaced by MDR1b expression during pregnancy. We have studied whether expression of MDR1b and CFTR also vary in the lung during pregnancy. No variations in MDR1b or CFTR mRNA levels during pregnancy were detected. However, there was an unusual degree of variation in MDR1b mRNA expression in lung parenchyma between animals in both the control group and the pregnant group. If present among humans, polymorphic expression of MDR1 in lung parenchyma may explain part of the differences in lung symptomatology observed in the CF patients carrying the same mutation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Colocalization of leptin receptor (OB-R) mRNA and placental lactogen-II in rat trophoblast cells: gestational profile of OB-R mRNA expression in placentae.

The present study was designed to clarify the cellular localization and expression of leptin receptor(s) [OB-R(s)] mRNA including its splice variants and their correlation with the cells which secrete placental hormone, placental lactogen-II (PL-II), in rat placentae. By in situ hybridization analysis, hybridization signals for OB-Rb and the common extracellular domain of OB-R were first detectable in some cells of the labyrinth zone of the placentae on day 14 of pregnancy and then a lot of cells dispersed in the entire area of the labyrinth zone expressed OB-Rb during the latter half of pregnancy. However, no expression was observed in the decidua and the junctional zone of the placentae during pregnancy. Double staining study revealed that signals for OB-R expressing trophoblast cells showed PL-II immunoreactivity in the labyrinth zone of the placentae. In Northern blot analysis, two bands (2.8 kb and 5.1 kb) of OB-R mRNA expression were observed in the placentae from day 17 to 21 of pregnancy and the expression of both increased markedly up to day 21 of pregnancy. RT-PCR analysis revealed that OB-Rb, OB-Ra, and OB-Re are expressed in the placentae on days 19 and 21 of pregnancy. These results suggest that the OB-R may have a physiological significance in the placental function during the latter half of pregnancy.

Alternative Splicing↗
PubMed1993

Co-existing structures of an mRNA stability determinant. The 5' region of the Escherichia coli and Serratia marcescens ompA mRNA.

The structure of untranslated regions of mRNA is thought to play a key role in the degradation of mRNAs by specific RNases. As a model system, in vitro transcripts of the stability determining 5' non-coding region of bacterial ompA mRNA were investigated by calculation of secondary structure models and by experiments applying the temperature-gradient gel electrophoresis (TGGE). For the theoretical prediction of secondary structures an algorithm was used, which yields the structure of lowest free energy as well as a large set of suboptimal structures. Three structures were predicted to co-exist in similar concentrations under native conditions. They denature in a low temperature transition leading to a unique structure which denatures in a high temperature transition. The prediction of three structures and two transitions could be confirmed experimentally by TGGE. Due to the use of transcripts of different length the conformational transitions could be attributed to distinct parts of the molecules. A pseudoknot structural motif was predicted theoretically, but could not be confirmed experimentally. Comparing ompA transcripts of E. coli and S. marcescens, a conservation of structural features could be shown in spite of a sequence homology of only 63%. Regarding the sequential folding of the transcript after synthesis, a metastable structure is formed first and is converted slowly into structures of lower free energy. The biological implications for in vivo degradation are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1996

Spatial segregation of G alpha s mRNA and vasopressin mRNA to distinct domains of the rough endoplasmic reticulum within secretory neurons of the rat hypothalamus.

The comparative subcellular localization of the mRNAs encoding the stimulatory subunit of heterotrimeric G-proteins (G alpha s) and the vasopressin-secreted peptide was performed at the light and electron microscopic level. We find that although the G alpha s membrane protein is devoid of signal peptide sequence at its N-terminal extremity, its mRNA is aggregated on defined domains of the rough endoplasmic reticulum (RER). This suggests that the G alpha s protein is probably synthesized close to the RER, and that, on the pathway to the plasma membrane, this protein might be primarily associated with RER membranes. We further find that the mRNAs encoding the G alpha s membrane-attached protein and the secreted peptide vasopressin have different patterns of distribution within the neuronal perikarya. Overall, our results show that these two mRNAs are segregated to distinct domains of the RER. We speculate that the RER might be organized in specialized domains involved in distinct functions with respect to mRNA translation and/or protein postranslational modifications.

Animals↗
PubMed1996

Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator.

Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA. It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6). In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA. Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs. Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV). However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6. Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element. Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA.

Caulimovirus↗