1. Volume 17, Issue 3, Pages 429-439 (February 2005)
Steroid Hormone Receptor Coactivation and Alternative RNA Splicing by U2AF65- Related Proteins CAPERα and CAPERβ 
Dennis H. Dowhan, Eugene P. Hong, Didier Auboeuf, Andrew P. Dennis, Michelle M. Wilson, Susan M. Berget, Bert W. O'Malley 
Molecular Cell 
Volume 17, Issue 3, Pages (February 2005)
DOI: /j.molcel

2. Figure 1
Sequence, Functional Domains, and RNA Expression Profile of CAPERβ
(A) The predicted amino acid sequence of CAPERβ. The RS domain is from amino acids 59 to 100 and is boxed. The two RRM domains of CAPERβ are underlined and are between amino acids 152–226 and amino acids 249–322.
(B) A schematic alignment of the human proteins of CAPERβ, CAPERα, U2AF65, and PUF60 with their putative functional domains indicated (not to scale). The RS and RRM domains of PUF60, U2AF65, and CAPERα are aligned with CAPERβ, and the percentage similarity of these domains in relation to CAPERβ is indicated. The human chromosomal location for each gene is also indicated.
(C) Northern Blot analysis of CAPERβ, CAPERα, U2AF65, PUF60, and β-actin in human tissues. The relative positions of the RNA markers (kb) for each blot are indicated. Abbreviations are: skeletal muscle, SK. Muscle; small intestine, Sm. Intest.; and peripheral blood leukocyte, P.B.L.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 In Vitro Interaction of CAPERα and CAPERβ with ERα and PR
(A) GST-CAPERα, GST-CAPERβ, or GST-U2AF65 proteins were tested by GST pulldown assay for their ability to interact with either 35S-radiolabeled ERα, PR, or TRβ in the presence or absence of either 10−9 M estradiol (E2), 10−8 M progesterone (Pg), 10−7 M 3,5,3′-Triiodo-L-thyronine (T3), or vehicle (ethanol).
(B) Recruitment of RNA splicing factor CAPERβ to the pS2 promoter by chromatin immunoprecipitation (ChIP) assays. The estradiol-mediated recruitment of CAPERβ to the pS2 promoter in MMTV-CAT T47D cells treated with hormone for 1 hr was determined by ChIP. Abbreviations are: preimmune serum, PIS; anti-CAPERβ antibody, anti-CAPERβ; anti-PUF60 antibody, anti-PUF60; and anti-U2AF65 antibody, anti-U2AF65.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 CAPERα and CAPERβ Coactivate Steroid Hormone Receptors
(A) CAPERα and CAPERβ coactivate PR transcriptional activity from a progesterone response element linked to a minimal promoter. HeLa cells were cotransfected with PRE-E1b-luciferase reporter along with expression vectors for PR alone or PR with CAPERα or CAPERβ with vehicle (ethanol) or 10−8 M Pg as indicated and were tested for luciferase activity (see the Experimental Procedures).
(B) CAPERα and CAPERβ coactivate PR transcriptional activity from the MMTV promoter. HeLa cells were cotransfected with MMTV-luciferase reporter as in (A).
(C) CAPERα and CAPERβ enhance the transcriptional activity of ERα. HeLa cells were cotransfected with ERE-E1b-luciferase reporter along with expression vectors for ERα alone or ERα plus CAPERα or CAPERβ with vehicle (ethanol) or 10−9 M E2.
(D) CAPERα or CAPERβ do not coactivate the thyroid hormone receptor. CV-1 cells were cotransfected with HDI-TK-luciferase reporter along with expression vectors for TRβ alone or TRβ plus CAPERα or CAPERβ with vehicle (ethanol) or with 10−7 M T3.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
CAPERα or CAPERβ Modulate Alternative Splicing of the CT/CGRP Minigene in a Steroid Hormone-Dependent Manner
(A) A schematic diagram of the CT/CGRP minigene. The CT/CGRP minigene encompassing exon 4 (CT) through exon 5 and 6 (CGRP) was placed downstream of the HSV, PRE, ERE, or MMTV promoters.
(B) The splicing assays were preformed by using HeLa cells and either the HSV-CT/CGRP or PRE-CT/CGRP minigenes. The graph indicates the average (±SD, n = 4) fold hormonal effect of the CGRP/CT ratio from the RNA products, with or without CAPERα or CAPERβ, by dividing the CGRP/CT ratio obtained in the presence of 10−8 M Pg by the CGRP/CT ratio obtained in the absence of hormone for each experiment. Although the intensity of the PCR products for the PRE-CT/CGRP without hormone was very low on the autoradiograph shown, phosphorimaging did allow for quantification of these RNA products.
(C) The change in alternative splicing of the PRE-CT/CGRP minigene upon addition of CAPERα or CAPERβ. The graph indicates the average (±SD, n = 4) fold change of the CGRP/CT ratio of PRE-CT/CGRP minigene, with or without CAPERα or CAPERβ from the experiment in (B). The fold CGRP/CT ratio was obtained by dividing the CGRP/CT ratio obtained in the presence of PR, 10−8 M Pg, and CAPERα or CAPERβ by the CGRP/CT ratio obtained from PR, 10−8 M Pg, and empty expression vector.
(D) The MMTV-CT/CGRP minigene is alternatively spliced by CAPERα and CAPERβ. The graph indicates the average (±SD, n = 4) fold change of the CGRP/CT ratio of the MMTV-CT/CGRP minigene, with or without CAPERα or CAPERβ.
(E) CAPERα and CAPERβ were able to alternatively splice the ERE-CT/CGRP minigene. The splicing assays were preformed by using HeLa cells and the ERE-CT/CGRP minigene. The graph indicates the average (±SD, n = 4) fold change of the CGRP/CT ratio of the ERE-CT/CGRP minigene, with either ERα or ERβ and with or without CAPERα or CAPERβ.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
U2AF65 and PUF60 Do Not Function as Progesterone Receptor Coactivators for Transcription or Splicing
(A) U2AF65 and PUF60 do not coactivate PR. The PRE-E1b-luciferase reporter gene was transfected in either the presence or absence of the protein expression vectors as indicated and under the conditions described in Figure 3.
(B) Alternative splicing of the CT/CGRP minigene by U2AF65 and PUF60 is not promoter or steroid receptor dependent. The HSV- and PRE-CT/CGRP reporter genes were transfected under the conditions described in Figure 4. The graph indicates the average (±SD, n = 3) fold change of the CGRP/CT ratio of the PRE-CT/CGRP minigene, with or without expression vectors for U2AF65 or PUF60. The fold CGRP/CT ratio was obtained by dividing the CGRP/CT ratio obtained in the presence of PR, 10−8 M Pg, and U2AF65 or PUF60 by the CGRP/CT ratio obtained from PR, 10−8 M Pg, and empty expression vector.
(C) Alternative splicing of the CT/CGRP minigene by the PR coactivator SRC-1. The PRE-CT/CGRP reporter gene was transfected in either the presence or absence of the protein expression vectors as indicated and under the conditions described in Figure 4. The graph indicates the average (±SD, n = 4) fold change of the CGRP/CT ratio of the PRE-CT/CGRP minigene, with or without expression vectors for CAPERα, CAPERβ, or SRC-1. The fold CGRP/CT ratio was obtained as per Figure 4C.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Point Mutations in CAPERβ Identify Distinct Domains Important for Transcriptional Coactivation and Alternative Splicing
(A) A schematic diagram of CAPERβ, and the corresponding mutations to conserved amino acids within the RNP-2 consensus amino acid sequences of the two RRM domains and in the carboxyl-terminal region of CAPERβ. The underlined residues highlighted in the CAPERβ diagram represent the amino acid substitutions present in the three mutants. The mutations to CAPERβ were: CAPERβM1: F153/L157 to A153/A157, CAPERβM2: Y250/L254 to A250/A254, and CAPERβM3: L353/L357 to A353/A357.
(B) The PRE-E1b-luciferase reporter gene was transfected in either the presence or absence of protein expression vectors as indicated and under the conditions described in Figure 3. The graph represents the effect on luciferase activity of the various proteins indicated.
(C) Splicing assay were preformed by using HeLa cells with the PRE-CT/CGRP minigene under the conditions described in Figure 4. The graph indicates the average (±SD, n = 3) fold change of the CGRP/CT ratio of the PRE-CT/CGRP minigene, with or without expression vectors. The fold CGRP/CT ratio was obtained as per Figure 4C.
(D) A schematic diagram of the major VEGF isoforms in T47D cells, encompassing exon 5 (E5), exon 6 (E6), exon 7 (E7), and exon 8 (E8) (not to scale).
(E) Effect of siRNA to CAPERα and CAPERβ on the Pg-inducible, alternatively spliced VEGF gene. T47D cells were transfected with or without siRNA as indicated and were harvested, total RNA was prepared, and VEGF splicing was assessed by RT-PCR as described (see the Experimental Procedures). The graph indicates the average (±SD, n = 3) fold change of the VEGF-121/VEGF-189 ratio of the endogenous VEGF mRNA transcripts, with or without cells transfected with control siRNA duplexes or pooled siRNA duplexes targeting either CAPERα or CAPERβ. The fold VEGF ratio was obtained by dividing the VEGF-121/VEGF-189 ratio obtained in the absence of 10−8 M Pg by cells treated with 10−8 M Pg and transfected with or without control siRNA duplexes or pooled siRNA duplexes targeting either CAPERα or CAPERβ. An autoradiograph of the radiolabeled VEGF, CAPERα, CAPERβ, and U2AF65 RT-PCR products from a representative experiment is shown. The CAPERβ RT-PCR was exposed three times longer than the autoradiographs for CAPERα and U2AF65.
Molecular Cell  , DOI: ( /j.molcel )