Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 is a regulator of both transcription and alternative splicing. MCF7 cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. RNA was extracted, libraries prepared, and RNA-seq was conducted. ( A ) Venn diagram representing differentially expressed genes (light green) and alternatively spliced genes (light orange). ( B , C ) Functional analysis was conducted using the DAVID functional annotation tool (DAVID, https://david.ncifcrf.gov/ ) for ( B ) differentially expressed genes and ( C ) alternative splicing genes. ( D ) Real-time PCR analysis of total mRNA amount of CD44 relative to CycloA reference gene. ( E ) Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE; (**) P < 0.01 (paired Student's t -test).

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing, Transfection, RNA Sequencing, Functional Assay, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Tethering p300 to CD44 promoter up-regulates its expression and promotes CD44v inclusion. ( A – C ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter, Intron 8 (In8) or 3′ UTR for 30 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44 relative to CycloA reference gene ( A ) and for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount ( B ). PSI was calculated by CD44v6-7/CD44s + CD44v6-7. ChIP was performed of H3 pan-acetylated, along the CD44 gene ( C ). Values represent averages of three independent experiments ± SE and are expressed as dCas9-p300 core (WT) relative to dCas9-p300 core (mut) (*) P < 0.05, (**) P < 0.01, for gRNA to promoter; (#) P < 0.05 gRNA to In8; (†††) P < 0.005 gRNA to 3′ UTR; Student's paired t -test. ( D , E ) HEK293T cells were transfected with either WT RNAPII or Slow RNAPII and after 24 h treated with α-amanitin for 24 h ( D ); or cells were treated with 6 µM CPT for 6 h ( E ). Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent mean ± SE of seven ( D ) or three ( E ) independent experiments.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 acetylates Sam68 and hnRNP M. ( A ) Enrichment of binding sites of six of CD44's known splicing factors in p300 regulated exons: SAM68, hnRNP M, hnRNP L, TRA2β, hnRNP F, hnRNP A1. Shown are the −lg ( P -values) enrichment of binding sites in skipped exons versus included ( right bars) and included versus skipped ( left bars). Dashed red line denotes significant P -value (corrected for multiple testing, Bonferroni correction). As can be seen, the binding sites of SAM68 and hnRNP M were found to be significantly enriched in the skipped exons. ( B , C ) HEK293T cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. Sam68 was immunoprecipitated and acetylated lysine (AK) was detected ( B ). (*) Heavy chain of Sam68 Ab. hnRNP M was immunoprecipitated and acetylated lysine (KA) was detected ( C ). ( D ) MCF7 cells were transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) or against Sam68 (siSam68) or hnRNP M (sihnRNP M) for 72 h. Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE. (*) P < 0.05; (***) P < 0.001; Student's paired t -test. ( E ) HEK293T transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) and transfected again after 48 h with either pcDNA3-empty vector or hnRNP M for 24 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent averages of three independent experiments ± SE.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 excludes hnRNP M from CD44 pre-mRNA and promotes cell motility. ( A ) RNA-IP of hnRNP M in HEK293T cells transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter. Real-time PCR analysis of CD44 intron 8. Values represent averages of three independent experiments ± SE. ( B ) hnRNP M was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. ( C ) HDAC1 was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. (*) Long exposure. ( D ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter for 30 h. ChIP was performed on HDAC1, along the CD44 gene. ( E ) HEK293T cells were treated with 10 µM TSA for 2 h. Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. ( F , G ) MCF7 cells stable for dCas9-p300 core (mut) or dCas9-p300 core (WT) were transfected with four gRNAs targeted to CD44 promoter or 3′ UTR for 30 h. Cells were seeded in 96-well plates and a scratch assay was conducted. Results were calculated for gRNA to promoter relative to 3′ UTR. Values represent averages of three independent experiments ± SE. ( G ) Western blot was conducted using the indicated antibodies.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Wound Healing Assay, Western Blot

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Schematic representation of the proposed mechanism. The chromatin factor p300 acetylates histones as well as splicing factors to regulate, respectively, transcription and alternative splicing.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing