Article Title: RNA binding to CBP stimulates histone acetylation and transcription
Figure Lengend Snippet: In vitro reconstitution of CBP RNA binding A) CBP domains and RNA binding prediction (BindN). Non-binding (green) and binding residues (red). Magnified sequence shows predicted RBR in CBP-HAT wt . B-C) In vitro pull down of B) eRNA-Klf6 s and C) eRNA-Med13l s . D) Quantification of RNA-pulldown data in B-C. n=3. E) In vitro pull down of eRNA-Klf6 s . F) Quantification of RNA-pulldown data in B-C. n=3. G-H) RNA EMSA of eRNAs using CBP-HAT wt . G) eRNA-Mdm2 s ; H) eRNA-Med13l s . I-J) Competition binding RNA EMSAs. Binding of 2nM 32 -P radiolabelled eRNA-Mdm2 to CBP-HAT wt (2000nM) was competed with: I) 0–20nM unlabelled eRNA-Mdm2; J) 1nM, 10nM and 20nM un-labeled eRNA-Mdm2 (RNA), dsDNA or ssDNA with the same sequence. K) RNA EMSA using: K) CBP-HAT delta-loop and eRNA-Mdm2; L) CBP-HAT mutant-loop and eRNA-Med13l. RNA was titrated with 0–8000nM CBP-HAT. (*) CBP-HAT wt (2000nM). M) RBR mediates RNA binding to FL-CBP in vivo . PAR-CLIP for GFP-tagged CBP wt , CBP delta-loop or CBP mutant-loop in MEFs was followed by RT-qPCR. Control lncRNA Malat-1 was not identified by PARalyzer v1.1. P -values from two-tailed Student’s t-test: *P
Article Snippet: Sequencing libraries for PAR-CLIP experiments were prepared using NEB next small RNA library set for Illumina sequencing (New England Biolabs) according to manufacturers instructions.
Techniques: In Vitro, RNA Binding Assay, Binding Assay, Sequencing, HAT Assay, Labeling, Mutagenesis, In Vivo, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test