Abstract
The transcription factor cAMP-response element modulator (CREM) protein, plays a major role in cAMP-responsive gene regulation. Biological consequences resulting from the transcriptional stimuli of CREM are dictated by the expression of multiple protein isoforms generated by extensive alternative splicing of its precursor mRNA. We have previously shown that alternative splicing enables the expression of the CREM gene to be "switched" within the human myometrium during pregnancy from the production of CREMtau(2alpha), a potent transcriptional activator to the synthesis of CREMalpha, a transcriptional repressor. Furthermore we have recently reported that this change in the expression of CREM spliced variants is likely to have important ramifications on the regulation of downstream cAMP-response element-responsive target genes involved in uterine activity during gestation. We have investigated the splicing factors involved in controlling the expression of myometrial CREM splice variants. Data presented here from transient transfections indicate that the switch in the synthesis of CREMtau(2)alpha to CREMalpha that occurs during pregnancy is regulated primarily by an SR protein family member, SRp40. We also show that expression of this splicing factor is tightly regulated in the myometrium during pregnancy. SRp40 regulates the splicing of CREM via its interactions with multiple ESE motifs present in the alternatively exons of CREM. In vitro splicing and electrophoretic mobility shift assays were employed to confirm the functionality of the SRp40-binding ESEs, thus providing a mechanistic explanation of how SRp40 regulates the switch in splicing from production of CREMtau(2)alpha to CREMalpha.
Highlights
Transcriptional activation orchestrated by the cAMP-response element-binding protein/cyclic AMP-response element modulator (CREM)3/activating transcription factor family of transcription factors and cAMP-response elements (CREs) represents an important mechanism of cAMP-responsive gene regulation [1,2,3]
Co-transfection of pcDNA3.1CREM-1 and pcDNA3.1CREM-2 with the pCG-SRp40 plasmid resulted in increased expression of CREM2␣ mRNA spliced variants, which contain the alternatively spliced exon G, as reflected in the intensity of the 615-bp band compared with the 426-bp band (Fig. 3A (i), lane 4)
Alternative splicing promotes a switch in the expression of CREM2␣, an activator, to CREM␣, a repressor, within the human myometrium throughout pregnancy and labor
Summary
Construction of CREM Minigenes—A CREM minigene, pcDNA3.1 CREM-1, was generated from human genomic DNA using CREM-specific PCR primers, containing specific restriction sites, for the individual exon/intron fragments of CREM to facilitate the sequential insertion of each fragment into the pcDNA3.1(Ϫ) expression vector (Invitrogen). These primers, detailed in TABLE ONE, amplify CREM exons E and F (which encodes the phosphorylation domains), exon G (encoding the second glutamine-rich trans-activation domain), and the downstream exon H, together with truncated versions of their adjacent intronic
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