CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing

Abstract

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.

FIG. 1.

CoAA-mediated effects on transcription and splicing are enhanced by TRBP. (A) Agarose gel-based analysis and quantitative analysis by real-time PCR of Pg-stimulated recruitment of TRBP to a stably integrated MMTV promoter by the ChIP assay (see Materials and Methods). (B) HeLa cells were plated on 12-well plates 24 h before transfection in a 5% stripped serum-based medium. Each condition was used in triplicate wells. Per well, 5 ng of PR and 300 ng of MMTV-luciferase reporter gene were transfected with increasing amounts of pcDNA3-CoAA expression vector in the absence or in the presence of pcDNA3-TRBP expression vector as indicated. The amount of transfected DNA was equilibrated using the pcDNA3-empty expression vector. The serum-free transfection medium was replaced 6 h after transfection by a 5% stripped serum-based medium containing Pg (10⁻⁸ M). After 24 h of incubation, transfected cells were harvested for the luciferase assay. The luciferase activities obtained under the different conditions were divided by the luciferase activity obtained in the control wells transfected only with the pcDNA3-empty expression vector (first column). The histogram represents the mean and standard deviation (SD) of three separate experiments. (C) The CD44 minigene gives rise to three spliced variants containing either the two variable exon cassettes, v4 and v5 (inclusion), none of these exons (skipping), or one exon inclusion product (v4). HeLa cells were transfected as described for panel B, except that the reporter gene used was MMTV-CD44. Cells were harvested using 1 ml of TRIzol for each 12-well triplicate before RNA extraction. After DNase treatment, radiolabeled primers were used to amplify CD44 RNA products (see Materials and Methods). Autoradiograms of the radiolabeled-PCR products obtained in a representative experiment are shown. The histogram shows the mean (and SD, n = 3) quantification of the CD44 skipping/inclusion ratio obtained in the presence of different amounts of CoAA and/or TRBP expression vectors, divided by the control skipping/inclusion ratio obtained in the presence of only the empty expression vector (first column). (D) The CT/CGRP minigene contains two polyadenylation sites (pA) in either exon 4, giving rise to the CT product, or exon 6, giving rise to the CGRP product. HeLa cells were transfected as described for panel B, except that the reporter gene used was MMTV-CT/CGRP. Autoradiograms of the radiolabeled PCR products obtained in a representative experiment are shown. The histogram shows the mean (and SD, n = 3) quantification of the CT/CGRP ratio obtained in the presence of different amounts of CoAA and/or TRBP expression vectors, divided by the control CT/CGRP ratio obtained in the presence of only the empty expression vector (first column).

FIG. 2.

CoAA mediates promoter-preferential effects on transcription and alternative splicing. (A) MMTV-, CMV-, or HSV-luciferase reporter genes were transfected in HeLa cells as described in Materials and Methods. The MMTV-luciferase reporter gene, transfected with PR, was activated by Pg. The reporter genes were transfected with 300 ng of pcDNA3-CoAA (CoAA), pcDNA3-p54nrb (p54), pcDNA3-hnRNPA1 (A1), or pcDNA3 empty expression vector. The histograms represent the luciferase activity obtained in presence of either CoAA, p54nrb, or hnRNPA1 divided by the control luciferase activity obtained in presence of the empty expression vector. The means and SD were calculated from three separate experiments. (B) The same conditions as described for panel A were used, except that the reporter genes were MMTV-CD44, CMV-CD44, or HSV-CD44. The histograms represent either the fold effect of CoAA and p54nrb on the skipping/inclusion ratio (open boxes) or the fold effect of hnRNPA1 on the v4/inclusion ratio (solid boxes). The fold effect was obtained by dividing the ratio obtained in the presence of the different protein expression vectors by the ratio obtained in the presence of the empty expression vector (ø). Means and SD were calculated from three separate experiments. Representative autoradiograms of radioactive low-cycle RT-PCR amplification of splicing products are shown at the bottom.

FIG. 3.

The RRMs of CoAA mediate transcriptional effects on CMV-luciferase reporter gene. (A) CoAA, hnRNPA1, and p54nrb contain two N-terminal RNA RRMs and a C-terminal auxiliary domain either rich in glycine (G) and tyrosine (Y) residues (CoAA and hnRNPA1) or rich in acidic and basic residues (A/B, p54nrb). The RRMs and the auxiliary domains of these proteins were exchanged as described in Materials and Methods and as illustrated on the right. (B) CMV- or HSV-luciferase reporter genes were transfected in either the presence or the absence of various protein expression vectors as indicated and under the conditions described for Fig. 2. The histograms represent the effect on luciferase activity of the various proteins as indicated. Means and standard deviations were obtained from at least three separate experiments. (C) CMV-luciferase and MMTV-luciferase were transfected with TRBP expression vector and different protein expression vectors as indicated. The histograms representing the averages of three separate experiments represent the luciferase activity obtained in the presence of the various proteins and TRBP divided by the luciferase activity obtained in the control wells transfected with TRBP alone.

FIG. 4.

The RRMs of CoAA prevent splicing effects on CMV-CD44 products. MMTV-CD44 (A) or CMV-CD44 (B) reporter genes were transfected, as described in Materials and Methods, with or without the protein expression vectors as indicated. The histograms represent the fold effect of the different proteins on either the skipping/inclusion ratio or the v4/inclusion ratio. The fold effect was obtained by dividing the ratio obtained in the presence of the various protein expression vectors by the ratio obtained in the presence of the empty expression vector (Ø). Means and standard deviations were calculated from three separate experiments. Representative autoradiograms of radioactive low-cycle RT-PCR amplification of splicing products are shown on the right.

FIG. 5.

CoAA-mediated splicing effects are inversely correlated with its transcriptional effects on steroid-regulated promoters. PRE-, ERE-, or MMTV-luciferase reporter genes or PRE-, ERE-, or MMTV-CD44 reporter genes were transfected with 5 ng of either PR, ERα, or ERβ per well and with 300 ng of CoAA expression vector or empty expression vector per well. The open boxes represent the fold effect of CoAA on luciferase activity, and the solid boxes represent the fold effect of CoAA on the CD44 skipping/inclusion ratio. Means and SD obtained from at least three separate experiments are shown.

FIG. 6.

The CMV promoter does not completely abrogate CoAA-mediated splicing effects on CMV-transcribed RNA. MMTV-CT/CGRP (A) or CMV-CT/CGRP (B) reporter genes were transfected, as described in Materials and Methods, with or without different protein expression vectors as indicated. The histograms represent the fold effect of the various proteins on the CT/CGRP ratio. The fold effect was obtained by dividing the ratio obtained in the presence of the various protein expression vectors by the ratio obtained in the presence of the empty expression vector (Ø). Means and standard deviations were calculated from three separate experiments. Representative autoradiograms of radioactive low-cycle RT-PCR amplification of splicing products are shown on the right.

FIG. 7.

Competitive recruitment of RNA-binding proteins by the promoter and the transcript. A transcriptional/splicing regulator, like CoAA could be recruited to target promoters through either DNA-protein or protein-protein interactions. Depending on the affinity of the regulator for the transcriptional complex and for the transcript synthesized from this promoter, the regulator will or will not be able to interact with the transcript, participating or not participating in the splicing regulation process. For instance, the engagement of CoAA through its RRMs in the CMV promoter regulation (“Non-permissive interaction”) could restrain the ability of CoAA to be engaged in splicing (promoter A), whereas the engagement of CoAA through its auxiliary domain in the PR-activated MMTV promoter regulation (“Permissive interaction”) could allow this engagement (promoter B). In the natural context of a promoter driving the synthesis of an unique transcript, several mechanisms could coexist to modulate this competition either by changing the “strength” of the engagement of a given regulator in the transcriptional complex or by changing the affinity of the regulator for the RNA transcript (see Discussion).