Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5′ exons of protein 4.1R gene by Shu-Ching Huang, Aeri Cho, Stephanie Norton, Eva S. Liu, Jennie Park, Anyu Zhou, Indira D. Munagala, Alexander C. Ou, Guang Yang, Amittha Wickrema, Tang K. Tang, and Edward J. Benz Blood Volume 114(19):4233-4242 November 5, 2009 ©2009 by American Society of Hematology

Analyses of first exon expression. Analyses of first exon expression. (A) Schematic representation of the splicing patterns from exon 1 to its downstream 3′ ss. p3′ss indicates proximal 3′ss; d3′ss, distal 3′ss. AUG-1 located within exon 2′. (B) First exon expression in human and mouse cell lines and tissues. A common anti-sense primer annealing to exon 2 and variable forward primers specific to each of the first exons were used for PCR on cDNA synthesized by use of a downstream exon 2 primer. The top panels are PCR; the bottom panels are Southern hybridization with exon 2′ as a probe (Ex2′). (C) First exon expression during cultured CD34+ erythroid differentiation by use of the same RT-PCR strategies. The top panels are PCR products; the bottom panels are Southern-blot hybridizations that use exon 2′ as a probe (Ex2′). (D) Real-time PCR analysis of ratios of 1A:1C expression during CD34+ differentiation. 0, 4, 8, 10, and 14 indicate days of differentiation. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

Cotranscriptional pre-mRNA splicing events detected by ChIP and ChRIP assays. Cotranscriptional pre-mRNA splicing events detected by ChIP and ChRIP assays. (A) Schematic diagram of ChIP (adapted with permission from Listerman et al36); antibodies against splicing factors (SF) pull down chromatin regions attached to splicing factors through the nascent RNA and pol II (pol). CBC indicates cap-binding complex. (B) 4.1R DNA sequences detected in ChIP assays by the use of anti-SF2/ASF Ab. (Top panel) the 4.1R gene sequences amplified by indicated primer sets comprises the regions spanning intron upstream of exon 2′ and exon 2 (In/Ex2), exon 2′ and exon 2 (Ex2′/Ex2), and within exon 2 (Ex2/Ex2). DNA regions upstream of 1A promoter were amplified with primer sets up1/up2 and up3/up4. (Bottom panel) PCR. (C) Schematic diagram of ChRIP (adapted with permission from Listerman et al36); antibodies against AcH4 pull down the nascent RNA attached to the active chromatin through pol II. (D) Analyses of 4.1R pre-mRNA transcripts detected in anti-AcH4 (active chromatin) or anti-H3K4me1 (silenced chromatin) antibody precipitates. (Top panel) the region of primer annealing for RT-PCR is region. (Middle panel) 4.1R pre-mRNA spanning the region between the intron upstream of exon 2′ and exon 2 detected in input lysate, anti-AcH4, or IgG precipitates in the presence or absence of RT reaction. (Bottom panel) 4.1R pre-mRNA spanning the same region detected in input lysate, anti-H3K4me1, or IgG precipitates in the presence or absence of RT reaction. (E) 4.1R mRNA detected in anti-AcH4 or anti-H3K4me1 precipitates. (Top panel) the primer at exon 17 was used for RT and amplified by primer sets located at exons 13 and 17. (Middle panel) amplified products with or without exon 16 detected in anti-AcH4 precipitates in the presence or absence of RT reaction. Input lysate and IgG precipitates served as controls. (Bottom panel) amplified products with or without exon 16 detected in anti-H3K4me1 precipitates in the presence or absence of RT reaction. Input lysate and IgG precipitates served as controls. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

Expression of exon 2′/2 under the control of CMV or its native promoter. Expression of exon 2′/2 under the control of CMV or its native promoter. (A) Minigene constructs under the control of a CMV promoter, containing exon 1 and its respective 200, 300, and 400 bp of downstream intronic sequences joined with exon 2′/2 and an equal length of its upstream intronic sequences. (B) Exon 2′ splicing patterns in minigene-transiently transfected or nontransfected MELC. Splicing products were analyzed for exon 2′ inclusion by RT-PCR by the use of a vector-specific primer for minigene or exon 2-specific primer for endogenous 4.1R. (Top panels) RT-PCR products. (Bottom panels) Southern blot with exon 2′ probe (Ex2′). Endo indicates MELC endogenous exon 2′/2 splicing pattern. (C) Exon 2′ splicing patterns in MELC transfected with minigene constructs under the control of their native promoters. RNAs were analyzed from either transiently (T) or stably (S) transfected MELCs. MELCs stably transfected with the respective minigenes under the control of CMV promoters (pCMV) or endogenous (endo) RNAs served as controls. (Top panels) RT-PCR products. (Middle panel) Southern blot with exon 2′ probe (Ex2′). (Bottom panels) Southern blot with exon 2 probe (Ex2). For each construct, 4 transfections were performed per experiment. Each experiment was repeated at least 3 times. Standard deviations are omitted in results that consistently have 0% or 100% of the same product in all 4 reproducible experiments. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

Inhibition of transcription elongation did not alter 1A or 1B exon 2′/2 splicing patterns. Inhibition of transcription elongation did not alter 1A or 1B exon 2′/2 splicing patterns. (A) Analyses of exon 2′/2 expression in MELCs in the presence of 0, 50, 100, and 150 μmol/L of DRB. Cells were treated with DRB for 24 hours and RNAs analyzed for exon 2′ expression by the use of a common anti-sense primer located in exon 2 and sense primer located either at exon 1A or 1B. Ex2′ indicates Southern blot using exon 2′ as a probe; Ex2, Southern blot using exon 2 as a probe. (B) Analyses of exon 2′/2 expression in native promoter-driven 1A and 1B minigene stably transfected MELCs in the presence of 0, 50, 100, and 150 μmol/L of DRB. RNA collected from cells treated with DRB for 24 hours were analyzed for exon 2′ expression. Ex2′ indicates Southern blot using exon 2′ as a probe; Ex2, Southern blot using exon 2 as a probe. For each construct, 4 stable lines were performed per experiment. Each experiment was repeated at least 3 times. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

SF2/ASF UV cross-linked to the sequences at the junction of exon 2′/2. SF2/ASF UV cross-linked to the sequences at the junction of exon 2′/2. (A) UV cross-linking templates consisting of the wild-type (WT) or mutated (Mu1, Mu2) junction of exon 2′/2 and its flanking sequences. (B) Purified SF2/ASF used in the cross-linking experiments. (C) WT or mutant transcripts cross-linked to HeLa nuclear extract or SF2/ASF. 32P-labeled transcripts were subjected to UV cross-linking by the use of indicated nuclear extracts or purified SF2/ASF in the presence of tRNA as a nonspecific competitor. NE indicates nuclear extracts. (D) For competition assay, 25-fold molar excess of unlabeled WT or Mu1 and Mu2 transcripts was added in binding reactions. − indicates that the probe was incubated in the absence of competitors. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

Depletion of SF2/ASF affects 3′ splice-site usages from exon 1B but not 1A. Depletion of SF2/ASF affects 3′ splice-site usages from exon 1B but not 1A. (A) Schematic of SF2/ASF structural organization and the regions targeted by SF2-shRNAs (sh84, sh584, and sh726). RRM indicates RNA recognition motif. RS, arginine/serine-rich domain. (B) The reduction of endogenous SF2/ASF expression by SF2-shRNAs and the effects on 3′ ss usage. (Top panel) RT-PCR analyses of exon 1A and 1B splicing patterns from RNA isolated from either control nonsilencing shRNA (Non) or SF2-shRNA–treated 1A or 1B minigene expressing MELC. (Middle panels) Southern blot hybridization with exon 2′ (Ex2′) or exon 2 (Ex2) probes. For each shRNA, 4 transductions were performed per experiment. Each experiment was repeated at least 3 times. Mean values ± SD of 3 independent experiments are shown. Standard deviations are omitted in results that consistently have 0% of exon 2′-inclusion product in all 3 reproducible experiments. (Bottom panel) equal amounts of cell lysates from control nonsilencing shRNA (Non)- and SF2-shRNA–treated cells were Western blotted for the presence of SF2/ASF. Expression, SF2/ASF expression levels in shRNA-treated cells relative to nonsilenced cells. β-actin served as loading control. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology

Recruitment model for cotranscriptional splicing of exon 2′/2. Recruitment model for cotranscriptional splicing of exon 2′/2. In this model, promoters 1A and 1B have differential abilities to bind SF2/ASF to the CTD of pol II. (A) In the 1A promoter, SF2/ASF is not recruited to the CTD; transcription of pre-mRNA exposes the distal 3′ss and allows for joining of exon 1A to produce a mature mRNA lacking exon 2′. (B) In the 1B promoter, SF2/ASF is recruited to the CTD of pol ll. The bound SF2/ASF blocks the distal 3′ss, favoring use of the proximal 3′ss, and results in the inclusion of exon 2′. Shu-Ching Huang et al. Blood 2009;114:4233-4242 ©2009 by American Society of Hematology