Journal: Cell reports

Article Title: RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery

doi: 10.1016/j.celrep.2019.08.022

Figure Lengend Snippet: (A and B) In vitro UV crosslinking analysis of indicated amount of recombinant CstF64 (A) or CFIm25 (B) protein bound to radiolabeled PAS2 oligonucleotide in the absence or presence of increasing amounts of recombinant CELF2 (left) or hnRNPL (right). hnRNPL protein crosslinked to M1 oligonucleotide is shown as a positive control for hnRNPL binding to RNA. The lane with hnRNP L alone was run on a different gel. (C) Left, schematic of reporter constructs and right, 3ʹ RACE analysis of mutant constructs upon transfection in HeLa cells with (+) or without (−) cotransfected CELF2 cDNA. Asterisk marks the product resulting from use of SV40 PAS. Red boxes indicate location of mutations that disrupt binding of CstF64, CFIm25, and CELF2, as indicated in the text. swPAS3 lacks all sequences around the native PAS3 (shown as a dark gray box), such that the vector-encoded non-PMA responsive SV40 polyA is the only other PAS present. See also .

Article Snippet: Recombinant CstF64 , Origene , TP304450.

Techniques: In Vitro, Recombinant, Positive Control, Binding Assay, Construct, Mutagenesis, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery

doi: 10.1016/j.celrep.2019.08.022

Figure Lengend Snippet: (A) Number of CELF2-regulated APA events that overlap with total activation-induced APA events, as detected by DaPars analysis of RNA-seq data from Jurkat cells. (B–E) 3′ RACE analysis of APA of the genes LRCH4 (B), RCCD1 (C), PNPO (D), and TNKS (E) in wild-type (WT) or CELF2-deficient (KD) cells before (−) or after (+) stimulation with PMA. For each panel, the gene name, 3′ UTR schematic, and RNA-seq tracks are shown from the indicated cell conditions. Proximal (P) and distal (D) PAS sites are indicated. CELF2 CLIP peaks from stimulated Jurkat cells (bottom bright red tracks), CFIm68 CLIP peaks from K562 cells (bottom yellow tracks), and CstF64 CLIP peaks from HepG2 cells (bottom gray tracks) are also shown. See for more examples. (F) Model showing CELF2 regulation of 3′ UTR IR and APA by competition with core processing machinery. Top: CELF2 promotes 3′ UTR IR by inhibiting U2AF65 binding to 3′ splice site. Bottom: CELF2 regulates APA by competing with core enhancer factors CFI and CstF upstream and downstream of PAS, respectively.

Article Snippet: Recombinant CstF64 , Origene , TP304450.

Techniques: Activation Assay, RNA Sequencing, Binding Assay

Journal: Cell reports

Article Title: RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery

doi: 10.1016/j.celrep.2019.08.022

Figure Lengend Snippet: (A) A scatterplot of stimulation-induced change in steady-state transcript level (log 2 [FC]) versus APA shifts (percentage distal polyA site usage index [PDUI]) for CELF2 target genes. Genes that also exhibit changes in their encoded protein (see B) are highlighted in red and labeled. (B) Abundance of proteins encoded by genes with CELF2 and/or activation-dependent APA (plus proteins known to change [CD69] or not [hnRNP L]) upon stimulation. Relative abundance is calculated from shotgun proteomics of Jurkat cells before and after stimulation, as described in the . Error bars represent SE from at least two independent experiments. (C) 3′ RACE analysis of APA of RBFOX2 in wild-type (WT) or CELF2-deficient (KD) cells without (−) or with (+) transfection of AMO targeting RBFOX2 distal site. 3′ UTR schematic and RNA-seq tracks are shown from the indicated cell conditions. Proximal(P) and distal (D) PAS sites are indicated. CELF2 CLIP peaks from stimulated Jurkat cells (bottom bright red tracks), CFIm68 CLIP peaks from K562 cells (bottom yellow tracks), and CstF64 CLIP peaks from HepG2 cells (bottom gray tracks) are also shown. Western blots show that inhibition of the distal PAS completely blocked the increase in RBFOX2 protein that is typically induced upon depletion of CELF2. hnRNPL is used as a loading control. AMO, antisense morpholino oligo.

Article Snippet: Recombinant CstF64 , Origene , TP304450.

Techniques: Labeling, Activation Assay, Transfection, RNA Sequencing, Western Blot, Inhibition, Control

Journal: Cell reports

Article Title: RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery

doi: 10.1016/j.celrep.2019.08.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant CstF64 , Origene , TP304450.

Techniques: Recombinant, Amplification, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Software