scholarly journals Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein GαsIs Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

2002 ◽  
Vol 277 (18) ◽  
pp. 15241-15251 ◽  
Author(s):  
Alison J. Pollard ◽  
Adrian R. Krainer ◽  
Stephen C. Robson ◽  
G. Nicholas Europe-Finner
1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.


1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001 ◽  
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.


2003 ◽  
Vol 94 (6) ◽  
pp. 2423-2432 ◽  
Author(s):  
Markéta Hrbasová ◽  
Jiri Novotny ◽  
Lucie Hejnová ◽  
František Kolář ◽  
Jan Neckář ◽  
...  

The present work has analyzed the consequences of chronic intermittent high-altitude hypoxia for functioning of the G protein-mediated adenylyl cyclase (AC) signaling system in the right (RV) and left ventricular (LV) myocardium in rats. Adaptation to hypoxia did not appreciably affect the number of β-adrenoceptors and the content of predominantly membrane-bound α-subunit (Gsα) of the stimulatory G protein, but it raised the amount of cytosolic Gsα in RV. The levels of myocardial inhibitory Gα protein were not altered. Activity of AC stimulated by GTP, fluoride, forskolin, or isoprotertenol was reduced by ∼50% in RV from chronically hypoxic rats, and a weaker depression was also found in LV. In addition, hypoxia significantly diminished a functional activity of membrane-bound Gsα in both RV and LV. The RV baseline contractile function was markedly increased in chronically hypoxic animals, and its sensitivity to β-adrenergic stimulation was decreased. Animals recovering from hypoxia for 5 wk still exhibited markedly elevated levels of cytosolic Gsα and significantly lower activity of AC in RV than did age-matched controls, but contractile responsiveness to β-agonists was normal.


1995 ◽  
Vol 268 (2) ◽  
pp. L221-L229 ◽  
Author(s):  
H. Kume ◽  
K. Mikawa ◽  
K. Takagi ◽  
M. I. Kotlikoff

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2445-2445
Author(s):  
Marie-Claude Gaudreau ◽  
Damien Grapton ◽  
Florian Heyd ◽  
Charles Vadnais ◽  
Brian T Wilhelm ◽  
...  

Abstract Hematopoiesis is sustained by a pool of multipotent hematopoietic stem cells (HSCs) that have the capacity to differentiate into cells of all blood cell lineages. The pool of long-lived HSCs is maintained throughout life by the self-renewal ability of HSCs. New evidence suggests the process of alternative splicing is an important regulator of the maturation and activation of blood and immune effector cells. It is presently estimated that almost all multi-exon genes in human genome undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. However, the role that pre-mRNA splicing may have for HSCs behaviour remains largely unexplored. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein that regulates alternative splicing by binding exonic splicing silencers elements (ESS) resulting in exon exclusion from the mature mRNA. RT-PCR analyses showed that hnRNPL is expressed in early stages of hematopoiesis including HSCs and lineage restricted hematopoietic progenitors. To test the role of hnRNPL in hematopoietic differentiation, we have generated conditional deficient mice, since a constitutive deletion of hnRNPL results in early embryonic lethality. Animals carrying two hnRNPL-floxed alleles (hnRNPLfl/fl) can be deleted at adult stage by the pIpC inducible MxCre transgene or by the VavCre transgene, which is expressed in all hematopoietic cells starting at embryonic stage E14. VavCre+hnRNPLfl/fl mice were not viable and did not progress further in their development than embryonic stage E17.5 and ablation of hnRNPL by pIpC injection caused a high rate of mortality in adult MxCre+hnRNPLfl/fl mice compared to control animals. Both the fetal liver (FL) of VavCre+hnRNPLfl/fl mice and the bone marrow (BM) of adult MxCre+hnRNPLfl/fl mice had a significantly reduced cellularity. Furthermore, flow cytometric analysis revealed in both FL and BM a significant reduction in frequency and absolute numbers of all mature blood cells, the lymphoid and myeloid precursors, CLPS, CMPs and GMPs and to a lesser extent the erythroid/megakaryocytic precursors (MEPs). Methylcellulose and both competitive and non-competitive transplantation assays demonstrated that HSCs lacking hnRNPL cannot generate lineage-committed progenitors and have lost their self-renewal capacity and reconstitution potential. A genome-wide analysis of mRNA expression and splicing through next-generation RNA sequencing of wild-type (WT) or VavCre+hnRNPLfl/fl E14.5 Lin- c-kit+ fetal liver cells (FLCs) revealed that hnRNPL deficiency affects not only alternative splicing but also gene expression levels in hematopoietic progenitors. In the absence of hnRNPL, genes implicated in regulating apoptosis, DNA damage response and cell division where found up-regulated in Lin- c-kit+ FLCs. Among those genes, many were p53 effector genes such as Cdkn1a, Ccng1, Trp53inp1, TrailR2, Bax and Zmat3. In addition genes that are known to be required for normal hematopoiesis and HSCs functions such as Gfi1, CD34, Csfr1, Egr1 and Runx1 were found down-regulated in those cells. Further analyses by qPCR and Western blots confirmed those findings and also showed that the level of p53 protein expression was upregulated in VavCre+hnRNPLfl/fl FLCs although the mRNA level is the same as in the WT cells suggesting that hnRNPL affects p53 mRNA translation efficiency. Similarly, several genes found differentially spliced are implicated in cell cycle progression or required for normal hematopoiesis in FL such as Bcl11a, Cdk4, Ccnd2 and TRP53bp1. These results together with an increased level of Reactive Oxygen Species (ROS) and elevated levels of phosphorylated histone H2AX (γ-H2AX, a sensor for double strand DNA breaks) suggest that hnRNPL regulates the activation of a p53 dependent DNA damage response pathway in hematopoietic stem cells. As a consequence loss of hnRNPL results in a loss of hematopoietic stem and progenitor cells. Our data also suggest that hnRNPL does not only regulate alternative splicing but also expression levels of a set of specific effector genes involved in HSC survival, proliferation, ultimately affecting self-renewal. Disclosures: No relevant conflicts of interest to declare.


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