We have shown that steroid hormones coordinately control gene transcriptional activity and splicing decisions in a promoter-dependent manner. Our hypothesis is that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity also directly participate in alternative RNA splicing decisions. To gain insight into the molecular mechanisms by which transcriptional coregulators could control splicing decisions, we focused our attention on a recently identified coactivator, CoAA. This heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein interacts with the transcriptional coregulator TRBP, a protein recruited to target promoters through interactions with activated nuclear receptors. Using transcriptional and splicing reporter genes driven by different promoters, we observed that CoAA mediates transcriptional and splicing effects in a promoter-preferential manner. We compared the activity of CoAA to the activity of other hnRNP-related proteins that, like CoAA, contain two N-terminal RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain and either have or have not been implicated in transcriptional control. By swapping either CoAA RRMs or the CoAA auxiliary domain with the corresponding domains of the proteins selected, we showed that depending on the promoter, the RRMs and the auxiliary domain of CoAA are differentially engaged in transcription. This contributes to the promoter-preferential effects mediated by CoAA on RNA splicing during the course of steroid hormone action.Regulation of gene expression is a complex process occurring in several steps, including transcription, splicing, transcript 5Ј-and 3Ј-end modification (capping and polyadenylation, respectively), transcript export, and stability. It is now accepted that these different steps are "mechanistically" coupled: transcription is coupled to capping, splicing, and polyadenylation; and splicing is coupled to capping, polyadenylation, and export (9,30,32,40,44). An emerging view of the coupling among gene expression machines indicates that proteins involved in early steps in the pathway can influence subsequent downstream steps (9,30,32). Transcriptional coregulators recruited to target gene promoters by DNA-binding transcriptional factors, including nuclear receptors, could play a major role in this regard (2, 13, 35, 37). Among the more than 100 coregulators identified to date that participate directly in transcriptional regulation, a subset of these proteins are structurally or functionally related to proteins involved in pre-mRNA processing (2, 11, 13, 14, 37).Functional principles for the "mechanical" coordination between the different steps of the gene expression process are emerging (7,12,38). Most human primary transcripts contain several exons separated by introns that are removed during the splicing reaction. Due to the presence of multiple splice sites, the RNA-splicing process can lead to the production of multiple mature transcripts. Alternative splicing is more a rule than an e...

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones. G ene expression regulation is a multistep process, including the synthesis of the pre-mRNA or transcription; the 5Ј and 3Ј end maturation of the transcript or capping and polyadenylation, respectively; the removal of the introns from the premRNA; and the export to the cytosol of the mRNA and its translation (1). Steroid hormones play a major role in the control of cellular fate and cellular homeostasis by modulating the expression of genes, the products of which are involved in cellular programs such as apoptosis, proliferation, differentiation, and in cellular metabolism (2, 3). Most of the studies of steroid hormone action focus on transcriptional effects that are mediated by the binding of steroid hormone receptors to hormone response elements localized in target gene promoters (4). Nevertheless, the biological consequences resulting from the modulation of the transcriptional activity of genes cannot be precisely predicted because of alternative splicing. Approximately 60% of human pre-mRNAs undergo an alternative splicing process that results in the synthesis from one gene of different mRNAs encoding different proteins having different biological actions (5). For instance, the products of many genes involved in apoptosis are alternatively spliced and this splicingcan result in the synthesis of isoforms that antagonize each other by having pro-vs. antiapoptosis effects (6). Therefore, the alternative splicing process can change considerably the biological consequences resulting from the transcriptional modulation of steroid hormone target genes and an important question to address is the mechanis...

No abstract

Lysyl oxidase (LOX) is a collagen crosslinking enzyme essential for the deposition of collagen. We investigated the mechanism of estrogen mediated increases of LOX expression in rat cardiac fibroblasts and the role of estrogen receptors (ER). Adult rat cardiac fibroblasts were treated with ER‐α (MPP) and ‐β (PHTPP) specific antagonists, and a general ER inhibitor (ICI 181780), then treated with estrogen for 48 hrs. LOX protein expression was analyzed by Western blot. LOX catalytic activity was measured by fluorometric assay of cell‐conditioned media. Estrogen (1 to 50 ng/ml) increased LOX protein expression (150–290% for active form and 210–310% for proLOX vs control; P<0.05). Estrogen also increased LOX activity in media (120–140% vs media from untreated). LOX activation by estrogen was prevented by ER‐α and ‐β blockade; however ER‐β antagonism had a greater effect, reducing LOX activity below that of control (75% versus control and ER‐α block; P<0.05). ER‐β blockade also had a greater effect on LOX protein expression (58% decrease for active LOX vs estrogen treated) than ER‐α inhibitor (25%). ICI also significantly decreased LOX protein expression. These data indicate that both ER‐α and ‐β are involved in estrogen induced upregulation of LOX expression and activity in rat cardiac fibroblasts, with ER‐β playing a more significant role (Louisiana Board of Regents LEQSF2009‐12‐RDA10 and AHA #11GRNT7700002).

No abstract