1. Volume 45, Issue 4, Pages 459-469 (February 2012)
Mediator Complex Regulates Alternative mRNA Processing via the MED23 Subunit 
Yan Huang, Wencheng Li, Xiao Yao, Qi-jiang Lin, Jing-wen Yin, Yan Liang, Monika Heiner, Bin Tian, Jingyi Hui, Gang Wang 
Molecular Cell 
Volume 45, Issue 4, Pages (February 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of MED23-Interacting Proteins
(A) Baculovirus-expressed His-Flag-MED23 was purified by Ni-NTA beads and analyzed by SDS-PAGE followed by Coomassie blue staining.
(B) Purified GST-E1A or its point mutant GST-H160Y was incubated with His-Flag-MED23 immobilized on the Ni-NTA beads at 4°C overnight. After washing, the bound proteins were eluted by boiling and immunoblotted with the anti-GST (for E1A) and anti-Flag (for MED23) antibodies. Input proteins were shown by Coomassie blue staining.
(C) Scheme for affinity purification and MS/MS to identify MED23-binding proteins.
(D) The protein profiles from the first elution (E1, after NTA affinity purification) and the second elution (E2, after M2 affinity purification) described in (C) were visualized by silver staining.
(E) Gene Ontology (GO) analysis of potential MED23-associated factors with or without RNase treatment.
(F) Venn diagram of MED23-associated proteins identified by affinity columns and MS/MS (with or without RNase treatment during purification).
(G) Venn diagrams of proteins identified by MS/MS following the affinity columns of MED23, in pair with those of MED24, MED16, MED15, and MED29, respectively.
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3. Figure 2
HnRNP L Specifically Interacts with MED23 In Vivo and In Vitro
(A) CoIP of hnRNP L with MED23. Flag-Med23-expressing plasmid was cotransfected with Myc-hnRNP L or Myc-hnRNP LL into 293T cells. Whole-cell extract was used for immunoprecipitation with the anti-Flag antibody, followed by western blotting using indicated antibodies.
(B) Soluble His-Flag-MED23 was incubated at 4°C overnight with immobilized GST-hnRNP L or GST-hnRNP LL. After washing, the bound proteins were eluted by boiling and immunoblotted with the indicated antibodies.
(C) Reciprocal coIP experiments with antibodies against endogenous hnRNP L and MED23 in HeLa nuclear extracts.
(D) Mediator complex purified from HeLa nuclear extract was subjected to SDS-PAGE, then analyzed by silver staining and western blotting. MED, Mediator fractions prepared through four columns; L-MED, Mediator fractions prepared through two columns (see the Supplemental Experimental Procedures for details).
(E) Nuclear extracts prepared from Med23+/+ (WT) or Med23−/− (KO) mouse embryonic stem cells were subjected to coIP using CDK8 antibody immobilized on protein G beads. The immunoprecipitated proteins were detected with indicated antibodies by western blotting.
(F) Deletion mutants of Flag-MED23 were coimmunoprecipitated with GST-hnRNP L using anti-Flag antibody. Asterisk indicates the IgG heavy chain.
(G) Myc-tagged hnRNP L deletion mutants were coimmunoprecipitated with Flag-MED23 using the anti-Flag antibody. Asterisks indicate the IgG heavy and light chains.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Association of MED23 with the Splicing Machinery
(A) HeLa nuclear extract was immunoprecipitated with anti-MED23 or control IgG. Total RNAs were extracted and separated on a denaturing polyacrylamide gel containing 8 M urea and detected by northern blot analysis with individual U snRNA probes. The immunoprecipitated proteins were immunoblotted with the indicated antibodies.
(B) HeLa cells were immunofluorescence stained with antibodies against MED23 and hnRNP L. The scatterplot of FITC and RRX emission intensities are plotted on the x and y axes, respectively, using Volocity Software. Coefficient for colocalization (Pearson correlation coefficient, r) was 0.44 ± 0.17 (see the Supplemental Experimental Procedures for details).
(C) HeLa cells were immunofluorescence stained with antibodies against MED23 and U1-70K. Colocalization was analyzed as in (B). r = 0.59 ± 0.06.
(D) HeLa cells were immunofluorescence stained with antibodies against MED23 and U2AF65. Colocalization was analyzed as in (B). r = 0.59 ± 0.13.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 MED23 Regulates Alternative Splicing
(A) Schematic representation of SLC2A2 minigene driven by CMV promoter. The alternatively spliced exon 4 is indicated by a gray box. Wild-type (wt) and substituted (sub) sequence elements are shown. Vertical lines within the introns mark the positions where intron sequences were deleted. Minigenes were transfected into the Med23+/+ (wild-type) or Med23−/− MEF cells, and splicing pattern was examined by RT-PCR. Control transfection (mock) was carried out in the absence of DNA. Average inclusion rates and standard deviations are from three separate experiments.
(B) CMV promoter-driven SLC2A2 minigenes were transfected into the si-Ctrl or si-Med23 HeLa cells, and splicing pattern was examined by RT-PCR. Average inclusion rates and standard deviations are from three separate experiments.
(C) CMV promoter-driven SLC2A2 minigene and increasing dosage of Flag-Med23 were transfected into HeLa cells, and splicing pattern was examined by RT-PCR. The expression of Flag-MED23 was detected by western blotting using Flag antibody.
(D) Schematic representation of CMV-promoter eNOS construct carrying 32 CA repeats in the shortened intron 13. The eNOS construct was transiently transfected into Med23+/+ (wild-type) or Med23−/− MEF cells, and splicing activity was determined by RT-PCR with exon 13/14-specific primers. The averages of splicing efficiencies with standard deviations are shown below the gel (n = 3).
(E) Schematic of RFXANK minigene with the CMV promoter is represented. Wild-type (wt) and substituted (sub) sequence elements are shown. Minigenes were transfected into the Med23+/+ (wild-type) or Med23−/− MEF cells, and splicing pattern was examined by RT-PCR. Average inclusion rates and standard deviations are from three separate experiments.
(F) Schematic of SLC2A2 minigene with the Egr1 promoter is represented. The Egr1 promoter-driven minigene was transfected into Med23+/+ (wild-type) MEF, Med23−/− MEF, or Med23−/− MEF with reexpressing MED23. Splicing pattern was examined by RT-PCR. Average inclusion rates and standard deviations are from three separate experiments.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Genome-wide Identification of AS and APA Events Commonly Targeted by MED23 and hnRNP L
(A) Knockdown of Med23 or hnRNP L in HeLa cells by retrovirus-mediated siRNA. Western blot was performed to detect the knockdown efficiency (left). Log2 (ratio) in the exon array data was shown, indicating the mRNA expression level of Med23 and hnRNP L in knockdown samples (right). Standard deviations are from three separate experiments.
(B) Scatterplot showing skipped exons in si-Med23 or si-hnRNP L cells as compared to si-Ctrl cells. y and x axes are splicing index (SI, see the Supplemental Experimental Procedures for detail). AS events regulated (p < 0.05, t test; and log2 (fold change) > 0.5) in si-Med23 only are in black, those regulated in both si-Med23 and si-hnRNP L are in red, and other skipped exons are shown in gray. Pearson correlation coefficient (r) and p value for the events shown in black and red are indicated.
(C) The numbers of AS events regulated in the same or opposite directions between si-Med23 and si-hnRNP L. Ex, exclusion; in, inclusion.
(D) As in (B), except that APA events are shown, and y and x axes are RUD values (see the Supplemental Experimental Procedures for detail).
(E) As in (C), except that APA events were analyzed. Le, 3′UTR lengthened (proximal poly[A] site less used); sh, 3′UTR shortened (proximal poly[A] site more used).
(F) Occupancy of hnRNP L on the set of genes whose AS events are coregulated by MED23 and hnRNP L in si-Ctrl (red) and si-Med23 (blue) HeLa cells, determined by ChIP-seq analysis. All these genes were normalized to 3 kb for metagene, with 1 kb extended upstream from TSS, and 1 kb downstream from TTS for analyzing average intensity in 50 bp bins.
(G) Quantitation of hnRNP L occupancy on promoter (1 kb upstream from TSS) and gene body region (from TSS to TTS). hnRNP L average binding intensity in si-Med23 (blue) was normalized to si-Ctrl (red).
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7. Figure 6 Transcriptional Control by MED23 and hnRNP L
(A) Heat map showing gene expression changes in si-Med23 or si-hnRNP L cells. Log2(ratio) for si-Med23 or si-hnRNP L versus si-Ctrl is represented by color using the scheme shown on the right. Only genes regulated in si-Med23 and/or si-hnRNP L are shown.
(B) Scatterplot showing regulation of gene expression in si-Med23 or si-hnRNP L cells as compared to si-Ctrl cells. Y and X axes are log2(ratio). Genes regulated (p < 0.05, t test; and fold change > 1.3) in si-Med23 only are in black, those regulated in both si-Med23 and si-hnRNP L are in red, and other genes are in gray. Pearson correlation coefficient (r) and its p value for genes shown in black and red are indicated. Experimentally validated genes (Figure S6B) are circled.
(C) Western blot detection of RNAi efficiency in Med23 or hnRNP L knockdown ESCs.
(D) Scheme shows primers used for real-time PCR of Egr1 gene locus.
(E–J) Egr1 mRNAs were detected by real-time PCR in si-L (E) or si-23 (F) HeLa cells. Standard deviations are from three separate experiments. Serum-stimulated ESCs were subjected to ChIP procedure using anti-Pol II antibody (G and H) or anti-H3K36me3 antibody (I and J) in si-L or si-23 HeLa cells.
(K) Model for the role of Mediator in coupling transcription and AS. The transcriptional Mediator cofactor complex communicates with the splicing machinery via interaction with splicing regulator.
Molecular Cell  , DOI: ( /j.molcel )
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