Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.
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PMID:Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. 1127 90

To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase-polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the beta deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma.
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PMID:Possible regulation of telomerase activity by transcription and alternative splicing of telomerase reverse transcriptase in human melanoma. 1140 73

1. Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABA(A) receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex. 2. In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 microM) did not significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 microM) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50-100 microM) was effective in enhancing GABA(A)-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; 1 or 10 microM), failed to depress GABA-induced currents in the cNTS. 3. Microinjections of midazolam (10 and 100 microM solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 microM) significantly and reversibly depressed it (gain decrease to 53 +/- 11 % of control, P < 0.01). 4. Reverse transcriptase polymerase chain reaction revealed the presence of alpha(1), alpha(2), beta(2), beta(3) and gamma(2) GABA(A) receptor subunit mRNA in the cNTS. No alternatively spliced variants of the alpha(1)- and gamma(2)-subunits were revealed. Moreover, GABA(A) epsilon-unit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts. 5. Immunocytochemical analysis revealed numerous GABA(A) epsilon-subunit-positive neurones within the cNTS with significantly fewer epsilon-subunit-positive cells in the rNTS. 6. As incorporation of the epsilon-subunit in recombinant GABA(A) receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of epsilon-subunit expression. This is the first demonstration of a possible physiological impact of the epsilon-subunit on native GABA(A) receptors.
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PMID:GABA(A) receptor epsilon-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat. 1169 72

Fabry disease is an inborn error of glycosphingolipid catabolism, resulting from deficient activity of lysosomal alpha-galactosidase A (alpha-Gal A). A rare alternative splicing that introduces a 57-nucleotide (nt) intronic sequence to the alpha-Gal A transcript from intron 4 of the gene has been identified. In addition, a novel midintronic base substitution that results in substantially increased alternative splicing has been identified in a patient with Fabry disease who has the cardiac variant phenotype. The sequence of the patient's intron 4 contains a single G-->A transversion at genomic nt 9331 (IVS4+919 G-->A ), located at the minus sign4 position of the 3' end of the intronic insertion (nts 9278--9334 in the genomic sequence). Minigene constructs containing the entire intron 4 sequence with G, A, C, or T at nt 9331 within an alpha-Gal A complementary DNA expression vector were prepared and expressed in COS-1 cells. Whereas transfection of the G or T minigenes transcribed predominantly normal-sized transcripts, the transfection of the A or C minigenes produced a large amount of the alternatively spliced transcript. These results suggest that the G-->A mutation, within an A/C-rich domain, results in increased recognition of the alternative splicing by an A/C-rich enhancer-type exonic splicing enhancer. The intronic mutation was not observed in 100 unrelated unaffected men but was present in 6 unrelated patients with cardiac Fabry disease. Reverse-transcriptase polymerase chain reaction of total RNA of various normal human tissues revealed that the alternatively spliced transcript was present in all of the samples, and especially at a higher ratio in the lung and muscle. The normal transcript was present in the patients' lymphoblasts and resulted in approximately 10% residual enzyme activity, leading to a cardiac phenotype of Fabry disease.
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PMID:Alternative splicing in the alpha-galactosidase A gene: increased exon inclusion results in the Fabry cardiac phenotype. 1182 41

Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.
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PMID:New alternatively spliced form of galectin-3, a member of the beta-galactoside-binding animal lectin family, contains a predicted transmembrane-spanning domain and a leucine zipper motif. 1188 49

The human ov-serpin monocyte neutrophil elastase inhibitor (MNEI) is encoded by a single gene SERPINB1. It is a highly efficient inhibitor of neutrophil granule proteases. Four murine genes with high sequence identity with MNEI were identified and fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB and EIC showed the same seven-exon gene structure as SERPINB1. However, EIC included an additional, alternatively spliced, exon due to the insertion of an endogenous retrovirus-like sequence. EID lacked several exons and is a pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is expressed at high levels in many tissues. EIB is mainly expressed in brain, and EIC was only expressed as splicing variants unlikely to encode a functional serpin. Upon incubation with serine proteases, EIA formed inhibitory covalent complexes with pancreatic and neutrophil elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB, EIC, and EID will be called Serpinb1, Serpinb1b, Serpinb1c, and Serpinb1-ps1. These data demonstrate that the four murine homologs of MNEI have met different evolutionary fates, and that EIA is the mouse ortholog of MNEI.
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PMID:Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1). 1218 54

Among the three G-protein-linked acetylcholine (ACh) receptors (GAR-1, -2, and -3) in Caenorhabditis elegans (C. elegans), GAR-3 appears most similar to mammalian muscarinic ACh receptors (mAChRs). The gar-3 gene, unlike mammalian mAChR genes, contains introns within the coding region. In this study, we identified an alternatively spliced isoform of GAR-3 (GAR-3a), which differs only in the third intracellular (i3) loop from the previously described one (GAR-3b). GAR-3a has a 26 amino acid insert in the i3 loop compared with GAR-3b. Reverse transcriptase-polymerase chain reaction (RT-PCR) on stage-specific RNAs indicated that both isoforms are expressed at all developmental stages examined, with gar-3b being more abundantly expressed than gar-3a. When these two GAR-3 isoforms were expressed in Chinese hamster ovary (CHO) cells, they exhibited similar ligand binding characteristics. In response to carbachol treatment, the two isoforms stimulated phosphatidylinositol hydrolysis with similar efficacy. Together with our earlier observations that GAR-1 and GAR-2 undergo alternative splicing, this study shows that alternative splicing plays an important role in promoting molecular diversity of G-protein-linked ACh receptors in C. elegans.
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PMID:Alternative splicing of the muscarinic acetylcholine receptor GAR-3 in Caenorhabditis elegans. 1292 13

The carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) plays an important role in coupling transcription with precursor messenger RNA (pre-mRNA) processing. Efficient capping, splicing, and 3'-end cleavage of pre-mRNA depend on the CTD. Moreover, specific processing factors are known to associate with this structure. The CTD is therefore thought to act as a platform that facilitates the assembly of complexes required for the processing of nascent transcripts. The mammalian CTD contains 52 tandemly repeated heptapeptides with the consensus sequence YSPTSPS. The C-terminal half of the mammalian CTD contains mostly repeats that diverge from this consensus sequence, whereas the N-terminal half contains mostly repeats that match the consensus sequence. Here, we demonstrate that 22 tandem repeats, from either the conserved or divergent halves of the CTD, are sufficient for approximate wild-type levels of transcription, splicing, and 3'-end cleavage of two different pre-mRNAs, one containing a constitutively spliced intron, and the other containing an intron that depends on an exon enhancer for efficient splicing. In contrast, each block of 22 repeats is not sufficient for efficient inclusion of an alternatively spliced exon in another pre-mRNA. In this case, a longer CTD is important for counteracting the negative effect of a splicing silencer element located within the alternative exon. Our results indicate that the length, rather than the composition of CTD repeats, can be the major determinant in efficient processing of different pre-mRNA substrates. However, the extent of this length requirement depends on specific sequence features within the pre-mRNA substrate.
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PMID:Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3'-end cleavage. 1503 67

The fibroblast growth factor receptor 2 (FGFR2) gene exons IIIb and IIIc are alternatively spliced in a mutually exclusive and cell type-specific manner. FGFR2 exon choice depends on both activation and silencing. Exon IIIb silencing requires cis-acting elements upstream and downstream of the exon. To examine the influence of transcription on exon IIIb silencing, the putative RNA polymerase II (RNAPII)-pausing MAZ4 element was inserted at different positions within the FGFR2 minigene construct. MAZ4 insertions 5' to the upstream silencing elements or between exon IIIb and downstream silencing elements result in decreased silencing. An insertion 3' of the downstream silencing elements, however, has no effect on splicing. An RT-PCR elongation assay shows that the MAZ4 site in these constructs is likely to be a RNAPII pause site. Insertion of another RNAPII pause site into the minigene has a similar effect on exon IIIb silencing. Transfection of in vitro transcribed RNA demonstrates that the cell type specificity of FGFR2 alternative splicing requires co-transcriptional splicing. Additionally, changing the promoter alters both FGFR2 minigene splicing and the MAZ4 effect. We propose that RNAPII pauses at the MAZ4 elements resulting in a change in the transcription elongation complex that influences alternative splicing decisions downstream.
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PMID:MAZ elements alter transcription elongation and silencing of the fibroblast growth factor receptor 2 exon IIIb. 1512 9

The thymosin beta 4 (Tbeta4) gene is of biological and pharmaceutical relevance because of its anti-inflammatory and wound-healing properties. As such, it is an example of a gene that may be targeted in immunotherapy regimens. Therefore, we have used the Tbeta4 gene to compare alternative strategies for RNA targeting, namely short hairpin (sh) RNAi versus external guide sequence (EGS)-mediated RNase P cleavage. Tbeta4 has two transcripts (UTbeta4 and LTbeta4) formed by alternative splicing that differ in both expression levels and the biological activity of their encoded products. Thus, we were able to compare the capacity of shRNAi/EGS mini-genes to target molecules of high and low abundance; to specifically target alternatively spliced mRNAs; and to discriminate between very closely related alleles encoding for identical proteins. Finally, we compared transient gene knockdown in tissue culture with results in stable systems in vitro and in vivo. The data demonstrate that shRNAi and EGS can both target the Tbeta4 gene, but that the extent of RNA reduction with shRNAi ( approximately 90%) is greater. RNAi targeting shows varying efficacy against two overlapping RNAs, is largely but not completely splice form-specific, and preferentially, but not exclusively, targets a perfect-sequence match. Very high targeting achieved with an shRNAi expressed from an RNA polymerase III promoter in transient transfection was not maintained in stably transfected clones and was not efficiently transmitted through the mouse germline. These results demonstrate the versatility and the limitations of RNA targeting strategies, and suggest that particular biological and clinical needs may be best met by varying the strategy.
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PMID:A comparative analysis of RNA targeting strategies in the thymosin beta 4 gene. 1535 35


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