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At least 235 records · Page 13Linked to original sources
PubMed2000

Tenotomy decreases reporter protein synthesis via the 3'-untranslated region of the beta-myosin heavy chain mRNA.

We tested the hypothesis that the beta-myosin heavy chain (beta-MHC) 3'-untranslated region (UTR) mediates decreased protein expression after tenotomy of the rat soleus. We also tested the hypothesis that decreased protein expression is the result of RNA-protein interactions within the 3'-UTR. beta-MHC was chosen for study because of its critical role in the function of postural muscles such as soleus. Adult rat soleus muscles were directly injected with luciferase (LUC) reporter constructs containing either the beta-MHC or SV40 3'-UTR. After 48 h of tenotomy, there was no significant effect on LUC expression in the SV40 3'-UTR group. In the beta-MHC 3'-UTR group, LUC expression was 37.3 +/- 4% (n = 5, P = 0.03) of that in sham controls. Gel mobility shift assays showed that a protein factor specifically interacts with the beta-MHC 3'-UTR and that tenotomy significantly increases the level of this interaction (25 +/- 7%, n = 5, P = 0.02). Thus the beta-MHC 3'-UTR is directly involved in decreased protein expression that is probably due to increased RNA-protein binding within the UTR.

3' Untranslated Regions↗
PubMed2003

Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.

Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD. However, the repetitive nature of the CTD prevents determination of phosphorylated sites by conventional biochemistry methods. Fortunately, a panel of monoclonal antibodies is available that distinguishes between phosphorylated isoforms of RNA polymerase II's (RNAP II) largest subunit. Here, we review how successful these tools have been in monitoring RNAP II phosphorylation changes in vivo by immunofluorescence, chromatin immunoprecipitation and immunoblotting experiments. The CTD phosphorylation pattern is precisely modified as RNAP II progresses along the genes and is involved in sequential recruitment of RNA processing factors. One of the most popular anti-phosphoCTD Igs, H5, has been proposed in several studies as a landmark of RNAP II molecules engaged in transcription. Finally, we discuss how global RNAP II phosphorylation changes are affected by the physiological context such as cell stress and embryonic development.

Amino Acid Sequence↗
PubMed2006

TRPC6 silencing in primary airway smooth muscle cells inhibits protein expression without affecting OAG-induced calcium entry.

TRPC proteins have been described as non-selective cation channels and are thought to be involved in the regulation of Ca(2+) movement in various cells, including airway smooth muscle (ASM) cells. In order to study the role of these channels in ASM cells, transfection of a small interfering RNA (siRNA) designed against the TRPC6 channel was performed in guinea pig primary ASM cells. This specific siRNA was complexed with the new X-TremeGene (X-TG) chemical transfection reagent, whose efficiency and low cytotoxicity were determined by the use of a non-silencing rhodamine-tagged siRNA. It was found that more than 95% of cells were transfected by an optimized protocol. Verification of TRPC6 transcript down-regulation was determined by RT-PCR while Western blot analysis attested to lower protein content in the microsomal fraction. Micro-spectrofluorimetry measurements of control and siRNA-treated cells revealed that lower TRPC6 expression did not affect OAG-induced intracellular Ca(2+) movement. Thus, TRPC6 channels cannot be defined as simple Ca(2+) transporters but more likely as protein complexes supporting monovalent cation conductance in ASM cells. These conductances would in turn facilitate membrane depolarization of high input resistance cells, Ca(2+) channel activation and tone increase. In conclusion, this study defines a valuable model of RNA interference study in primary cultures of ASM cells, eventually allowing for silencing of other target proteins for which no pharmacological modulators are currently available.

Animals↗
PubMed1999

Controlling human immunodeficiency virus type 1 gene expression by unnatural peptides.

Small unnatural peptides that target specific RNA structures have the potential to control biological processes. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of human immunodeficiency virus type 1 (HIV-1) gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all nascent HIV-1 transcripts. We report here a synthetic peptide derived from Tat sequence (37-72), containing all D-amino acids, that binds in the major groove of TAR RNA and interferes with transcriptional activation by Tat protein in vitro and in HeLa cells. Our results indicate that unnatural peptides can inhibit the transcription of specific genes regulated by RNA-protein interactions.

Amino Acid Sequence↗
PubMed2001

Expression of estrogen receptor isoforms alpha and beta messenger RNA in vaginal tissue of premenopausal and postmenopausal women.

OBJECTIVE: To determine the messenger RNA expression patterns of estrogen receptor (ER)alpha and ER beta in human vaginal tissue. STUDY DESIGN: Reverse transcriptase-polymerase chain reaction was performed on tissue samples of 75 patients having anterior colporrhaphy (25 premenopausal, 25 postmenopausal receiving estrogen replacement therapy [ERT], 25 postmenopausal not receiving ERT). Levels of mRNA were normalized and ratios were calculated to assess relative levels of expression. RESULTS: All samples showed expression of the ER alpha isoform. Significant differences existed in ER alpha expression among the 3 cohorts (P =.023). Greater differences (P <.001) existed in ER beta expression. For both isoforms, the premenopausal group had the highest level, and the postmenopausal group receiving ERT had the lowest level. No significant difference in ER beta expression existed between postmenopausal groups. CONCLUSION: Significant differences exist between premenopausal and postmenopausal women in presence and expression of ER alpha and ER beta in vaginal tissue. Expression of ER beta markedly declines in menopause, regardless of ERT use.

Adult↗
PubMed2005

The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2.

In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.

Adult↗
PubMed2000

High background levels compromise the use of cardiac troponin I RNA detection in peripheral blood as a diagnostic tool in cardiology.

The detection of cardiac troponins in peripheral blood as protein markers of myocardial infarction is a new diagnostic tool in the diagnosis of cardiac disease. In order to increase the sensitivity and specificity of this diagnostic approach, a reverse transcription polymerase chain reaction assay has been developed to detect the mRNA encoding cardiac troponin I from myocardial cells hypothetically released from damaged cardiac tissue. The detection is specific for cardiac troponin I mRNA, with no amplification of homologous sequences of other troponin I isoforms, i.e., troponin I from skeletal muscle cells. However, a strong amplification signal for cardiac troponin I mRNA was detected in samples of peripheral blood from healthy human volunteers. In patients with acute myocardial infarction or angina pectoris, the cardiac troponin I mRNA levels were not increased over background levels. In conclusion, a reverse transcription polymerase chain reaction approach based on the amplification of cardiac troponin I mRNA is not feasible in the diagnosis of cardiac diseases.

Angina Pectoris↗
PubMed2002

Distribution of alpha-1 adrenoceptor subtypes in RNA and protein in rabbit eyes.

We investigated subtypes of alpha-1 adrenoceptor (AR) in rabbit ocular tissues using reverse transcription-polymerase chain reaction (RT - PCR), in situ hybridization (ISH) and binding studies. Competitive RT - PCR assays specific for the subtypes of alpha-1 AR revealed that the mRNA expression of alpha-1a AR was dominant, and that of each alpha-1b and alpha-1d was less than 10% and 0.5% of total alpha-1 ARs mRNA, respectively, in the iris, ciliary body, choroid and retina. In alpha-1a AR splice isoform-specific RT - PCR assays, we found a distinct proportion of each isoform mRNA in the iris, ciliary body and choroid. The results of the ISH assays for alpha-1a AR subtype showed that hybridization signals were clearly observed in the iris dilator muscle and in the epithelium of the ciliary processes. In binding studies, alpha-1A AR was a dominant subtype in the iris, choroid and retina in contrast to the ciliary body that had more alpha-1B than alpha-1A AR subtype at protein level.

Adrenergic alpha-Antagonists↗
PubMed2005

Expression of icb-1 gene is interferon-gamma inducible in breast and ovarian cancer cell lines and affects the IFN gamma-response of SK-OV-3 ovarian cancer cells.

Icb-1 (C1orf38) is a human gene initially described to be involved in in vitro differentiation processes of endometrial adenocarcinoma and leukemia cells. In this study, we examined the effect of interferon-gamma on icb-1alpha and beta mRNA levels in human cell lines derived from breast cancer and gynecological malignancies. Furthermore, we intended to approach icb-1 gene function by means of RNA interference (RNAi) to analyze the effect of an icb-1 knockdown on human cancer cells in vitro. Three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231), three ovarian cancer cell lines (SK-OV-3, OVCAR-3 and BG-1) and the endometrial adenocarcinoma cell line HEC-1-A were treated with interferon gamma and the transcript levels of icb-1 isoforms alpha and beta were assessed by means of semiquantitative real-time RT-PCR. Our data demonstrates an interferon-gamma triggered upregulation of icb-1alpha mRNA levels in all breast cancer cell lines and an increase of icb-1beta mRNA in MDA-MB-231 cells. The strongest (about 10-fold) increase of icb-1alpha and beta mRNA after treatment with interferon-gamma was observed in ovarian cancer cell line SK-OV-3. Additionally, our data demonstrates the success of a siRNA-mediated knockdown of icb-1alpha and beta mRNA levels, which resulted in a significant increase of the antiproliferative interferon-gamma effect on SK-OV-3 cells. In conclusion, we report identification of the novel interferon-gamma inducible gene icb-1 which is able to affect the response of ovarian cancer cells to this cytokine.

Breast Neoplasms↗
PubMed2004

Neuregulin induces the expression of transcription factors and myosin heavy chains typical of muscle spindles in cultured human muscle.

Neuregulin (NRG) (also known as ARIA, GGF, and other names) is a heparin sulfate proteoglycan secreted into the neuromuscular junction by innervating motor and sensory neurons. An integral part of synapse formation, we have analyzed NRG-induced changes in gene expression over 48 h in primary human myotubes. We show that in addition to increasing the expression of acetylcholine receptors on the myotube surface, NRG treatment results in a transient increase of several members of the early growth response (Egr) family of transcription factors. Three Egrs, Egr1, -2, and -3, are induced within the first hour of NRG treatment, with Egr1 and -3 RNA levels showing the most significant increases of approximately 9- and 16-fold, respectively. Also noted was a corresponding increase in protein levels for both of these transcription factors. Previous literature indicates that Egr3 expression is required for the formation of muscle spindle fibers, sensory organs that are distinct from skeletal muscle contractile fibers. At the molecular level, muscle spindle fibers express a unique subset of myosin heavy chains. Two isoforms of the myosin heavy chain, the slow development and neonatal, were found to be increased in our myotube cultures after 48 h of treatment with NRG. Taken together, these results indicate that not only can NRG induce the expression of a transcription factor key to spindle fiber development (Egr3), but that a portion of this developmental process can be replicated in vitro.

Agrin↗
PubMed2006

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis.

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Actins↗
PubMed2000

Novel SCAMPs lacking NPF repeats: ubiquitous and synaptic vesicle-specific forms implicate SCAMPs in multiple membrane-trafficking functions.

In vertebrates, secretory carrier membrane proteins (SCAMPs) 1-3 constitute a family of putative membrane-trafficking proteins composed of cytoplasmic N-terminal sequences with NPF repeats, four central transmembrane regions (TMRs), and a cytoplasmic tail. SCAMPs probably function in endocytosis by recruiting EH-domain proteins to the N-terminal NPF repeats but may have additional functions mediated by their other sequences. We now demonstrate that SCAMPs form a much larger and more heterogeneous protein family than envisioned previously, with an evolutionary conservation extending to invertebrates and plants. Two novel vertebrate SCAMPs (SCAMPs 4 and 5), single SCAMP genes in Caenorhabditis elegans and Drosophila melanogaster, and multiple SCAMPs in Arabidopsis thaliana were identified. Interestingly, the novel SCAMPs 4 and 5 lack the N-terminal NPF repeats that are highly conserved in all other SCAMPs. RNA and Western blotting experiments showed that SCAMPs 1-4 are ubiquitously coexpressed, whereas SCAMP 5 is only detectable in brain where it is expressed late in development coincident with the elaboration of mature synapses. Immunocytochemistry revealed that SCAMP 5 exhibits a synaptic localization, and subcellular fractionations demonstrated that SCAMP 5 is highly enriched in synaptic vesicles. Our studies characterize SCAMPs as a heterogeneous family of putative trafficking proteins composed of three isoforms that are primarily synthesized outside of neurons (SCAMPs 2-4), one isoform that is ubiquitously expressed but highly concentrated on synaptic vesicles (SCAMP 1), and one brain-specific isoform primarily localized to synaptic vesicles (SCAMP 5). The conservation of the TMRs in all SCAMPs with the variable presence of N-terminal NPF repeats suggests that in addition to the role of some SCAMPs in endocytosis mediated by their NPF repeats, all SCAMPs perform a "core" function in membrane traffic mediated by their TMRs.

Animals↗
PubMed2001

A systematic gene expression screen of Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic inducible.

The soil nematode Caenorhabditis elegans is one of the simplest animals having the status of a laboratory model. Its genome contains 80 cytochrome P450 genes (CYP). In order to study CYP gene expression in C. elegans mixed stages and synchronized hermaphrodites were exposed to 18 known xenobiotic cytochrome P450 inducers. Messenger RNA expression was detected by DNA arrays and semiquantitative RT-PCR. Using subfamily-specific primers, a pooled set of exon-rich CYP fragments could be amplified. In this way it was possible to systematically check the influence of different inducers on CYP expression at the same time. The well-known CYP1A inducers beta-naphthoflavone, PCB52, and lansoprazol were the most active and in particular they strongly induced almost all CYP35 isoforms. A few number of further CYP forms were found to be inducible by other xenobiotics like phenobarbital, atrazine, and clofibrate. In addition, a transgenic C. elegans line expressing GFP under control of the CYP35A2 promoter showed a strong induction of the fusion by beta-naphthoflavone in the intestine.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

RNA content differs in slow and fast muscle fibers: implications for interpretation of changes in muscle gene expression.

Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression. However, total RNA content per gram of a particular fiber type may differ as well. We have tested this possibility in the distinct fiber types of adult rat skeletal muscle. Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content. Quantification of the hybridization showed that the 28S rRNA content decreases in the order I>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type IIB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per microgram of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis. Consequently, Type I fibers contain five- to sixfold more MHC mRNA per microgram of tissue than IIB fibers. These differences are not reflected in the total fiber protein content. This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition.

Animals↗
PubMed1999

Isotype-specific changes in the amount of beta-tubulin RNA in synchronized tobacco BY2 cells.

The 3'-ends of the beta-tubulin cDNA were amplified from tobacco BY2 polyA+ RNA. According to the differences in the predicted amino acid sequence at the extreme C-terminal, they were grouped into three different isotypes, NTB1 in which "EEGDYYEEDEEDLNEA", NTB2 in which "EEEYYEDEEEA QED" and NTB3 in which "DECEYEEEEEYDHEGN" follows the conservative "YQQYQDATAD" sequence. Using unique 3'-untranslated regions as probes, changes in the RNA levels of each beta-tubulin isotype were determined by dot-blot hybridization. The levels exhibited characteristic rhythms in the cell cycle. NTB1 RNA was highest in S phase in comparison to NTB2 RNA level which was highest in late G2. On the other hand, NTB3 RNA level was highest in early G2.

3' Untranslated Regions↗
PubMed2003

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron.

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5' cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.

Autoantigens↗
PubMed2004

Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel.

The persistent tetrodotoxin (TTX)-sensitive sodium current, detected in neurons of many regions of mammalian brains, is associated with many essential neuronal activities, including boosting of excitatory synaptic inputs, acceleration of firing rates, and promotion of oscillatory neuronal activities. However, the origin and molecular basis of the persistent current have remained controversial for decades. Here, we provide direct evidence that U-to-C RNA editing of an insect sodium channel transcript generates a sodium channel with a persistent current. We detected a persistent TTX-sensitive current in a splice variant of the cockroach sodium channel gene BgNa(v) (formerly para(CSMA)). Site-directed mutagenesis experiments revealed that an F-to-S change at the C-terminal domain of this variant was responsible for the persistent current. We demonstrated that this F-to-S change was the result of a U-to-C RNA editing event, which also occurred in the Drosophila para sodium channel transcript. Our work provides direct support for the hypothesis that posttranscriptional modification of a conventional transient sodium channel produces a persistent TTX-sensitive sodium channel.

Animals↗
PubMed2005

The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II.

Inactivation of the HRPT2 tumor suppressor gene is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumor syndrome and malignancy in sporadic parathyroid tumors. The cellular function of the HPRT2 gene product, parafibromin, has not been defined yet. Here we show that parafibromin physically interacts with human orthologs of yeast Paf1 complex components, including PAF1, LEO1, and CTR9, that are involved in transcription elongation and 3' end processing. It also associates with modified forms of the large subunit of RNA polymerase II, in particular those phosphorylated on serine 5 or 2 within the carboxy-terminal domain, that are important for the coordinate recruitment of transcription elongation and RNA processing machineries during the transcription cycle. These interactions depend on a C-terminal domain of parafibromin, which is deleted in ca. 80% of clinically relevant mutations. Finally, RNAi-induced downregulation of parafibromin promotes entry into S phase, implying a role for parafibromin as an inhibitor of cell cycle progression. Taken together, these findings link the tumor suppressor parafibromin to the transcription elongation and RNA processing pathway as a PAF1 complex- and RNA polymerase II-bound protein. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary parathyroid cancer.

Animals↗