Expression of human myometrial Gαs messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways

1997 ◽  
Vol 18 (1) ◽  
pp. 15-25 ◽  
Author(s):  
G N Europe-Finner ◽  
S Phaneuf ◽  
E Cartwright ◽  
H J Mardon ◽  
A López Bernal
Keyword(s):  
Alternative Splicing ◽  
Splice Variants ◽  
Protein Isoforms ◽  
Mrna Levels ◽  
Primary Role ◽  
Serine Residue ◽  
Rnase Protection ◽  
Mrna Splice ◽  
Messenger Ribonucleic Acid ◽  
Precursor Mrna

ABSTRACT We have shown previously that expression of 46 and 54 kDa human myometrial Gαs protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by Gαs-Small (with a serine residue at position 72) and Gαs-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a Gαs cDNA template to generate [32P]cytidine cRNA ribo-probes for use in RNase protection assays, so as to measure total myometrial Gαs mRNA levels in relation to the pattern of expression of Gαs mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial Gαs mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of Gαs precursor mRNA has a primary role in regulating expression of Gαs protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in Gαs mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Spatio-Temporal Expression of the Trans-Acting Splicing Factors SF2/ASF and Heterogeneous Ribonuclear Proteins A1/A1B in the Myometrium of the Pregnant Human Uterus: A Molecular Mechanism for Regulating Regional Protein Isoform Expression in Vivo*

2000 ◽  
Vol 85 (5) ◽  
pp. 1928-1936
Author(s):  
Alison J. Pollard ◽  
Colette Sparey ◽  
Stephen C. Robson ◽  
Adrian R. Krainer ◽  
G. Nicholas Europe-Finner
Keyword(s):  
Specific Protein ◽  
Protein Isoforms ◽  
Primary Role ◽  
Hnrnp A1 ◽  
Protein Species ◽  
Opposite Pattern ◽  
Messenger Ribonucleic Acid ◽  
Splicing Regulators ◽  
Spatio Temporal

Abstract Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.

Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53

Genome ◽  
2007 ◽  
Vol 50 (10) ◽  
pp. 946-953 ◽  
Author(s):  
Kim Disher ◽  
Adonis Skandalis
Keyword(s):  
Oxidative Stress ◽  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Splice Variants ◽  
Dna Lesions ◽  
Mrna Splicing ◽  
Aberrant Splice ◽  
Biological Phenomenon ◽  
Mrna Splice ◽  
Human Genes

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.

scholarly journals Identification of genes whose mRNAs are subjected to alternative splicing by endonuclease EndoG action in human and murine CD4+ T lymphocytes

2020 ◽  
Vol 3 (2) ◽  
pp. e00128
Author(s):  
D.D. Zhdanov ◽  
N.S. Novachly ◽  
M.V. Pokrovskaya ◽  
S.S. Aleksandrova ◽  
T.A. Kabardokov ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
T Lymphocytes ◽  
Splice Variants ◽  
Total Cell ◽  
Dna Damages ◽  
Mrna Splice ◽  
Healthy Humans ◽  
Number Of Cells

The aim of this work was to identify genes whose mRNAs were subjected to alternative splicing by apoptotic endonuclease EndoG in CD4+ T lymphocytes from healthy humans, mice, and rats. In order to induce EndoG, lymphocytes were transfected with an EndoG-containing plasmid, or a control pGFP plasmid, or were incubated with cisplatin. Efficiency of transfection, number of cells with DNA damages and the level of EndoG expression have been monitored. Total cell mRNA has been sequenced and the changes in proportion of splice variants of genes were analyzed. The changes in the proportion of 28 mRNA splice variants have been identified in human and murine lymphocytes in both transfected with EndoG gene or incubated with cisplatin. Thus, EndoG can be considered as a potent modulator of alternative splicing of mRNA of identified genes.

scholarly journals Reduced Parathyroid Vitamin D Receptor Messenger Ribonucleic Acid Levels in Primary and Secondary Hyperparathyroidism*

2000 ◽  
Vol 85 (5) ◽  
pp. 2000-2003
Author(s):  
Tobias Carling ◽  
Jonas Rastad ◽  
Eva Szabó ◽  
Gunnar Westin ◽  
Göran Åkerström
Keyword(s):  
Vitamin D ◽  
Parathyroid Adenoma ◽  
Messenger Rna ◽  
Hormone Secretion ◽  
Rnase Protection Assay ◽  
Mrna Levels ◽  
Vdr Gene ◽  
Rnase Protection ◽  
Messenger Ribonucleic Acid ◽  
Increased Risk

Abstract Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 ± 2.8% and 44 ± 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20–58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.

Regulation of mouse beta 3-adrenergic receptor gene expression and mRNA splice variants in adipocytes

1995 ◽  
Vol 268 (4) ◽  
pp. C1040-C1044 ◽  
Author(s):  
J. G. Granneman ◽  
K. N. Lahners
Keyword(s):  
Actinomycin D ◽  
Splice Variants ◽  
Receptor Gene ◽  
Mrna Translation ◽  
Mrna Levels ◽  
Mrna Splice ◽  
Receptor Mrna ◽  
Splicing Pattern ◽  
Total Beta

This study examined the regulation of murine beta 3-receptor mRNA and determined whether the recently described mRNA splice variants are differentially regulated by agents that alter total beta 3-receptor mRNA levels. In vivo treatment of mice with the beta 3-receptor agonist BRL-26830 reduced total beta 3-transcripts by 64% in white adipose tissue but did not alter the mRNA splicing pattern. Further analysis in cultured 3T3-F442A adipocytes showed that isoproterenol, dexamethasone, or phorbol 12-myristate 13-acetate also greatly reduced beta 3-receptor mRNA levels without selectively altering poly-U-containing transcripts. Blockade of transcription with actinomycin D produced a rapid loss of beta 3-receptor mRNA, which was prevented by blockade of mRNA translation with cycloheximide. However, neither actinomycin D nor cycloheximide altered the splicing pattern of beta 3-receptor mRNA. Analysis of transcription rate by nuclear run-off assay indicated that 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol 12-myristate 13-acetate reduce beta 3-receptor gene transcription and that suppression of transcription is sufficient to account for the reduction in beta 3-receptor mRNA levels by these agents.

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