Fig 1: HnRNP C regulates exon 3 inclusion independently of the GGGG motif and the poly-U tracts.(A) Western blot demonstrating the knockdown of hnRNP C using three different siRNA duplexes (siC1, siC2, siC3). Protein bands shown are from different lanes but within the same membrane. (B) Real-time RT-PCR analysis of RNA from K562 cells nucleofected with either control or hnRNP C-specific siRNA duplexes to assess the ratio of endogenous exon 3- to exon 4-containing BIM transcripts. “U” represents control cells that were not subjected to nucleofection. Results are presented as an average of three biological replicates and the relative endogenous E3: E4 ratio was determined by normalizing to the endogenous E3: E4 ratio of K562 cells that were not nucleofected with siRNA. Error bars represent ± SEM. *p<0.05, **p<0.01. (C) Schematic diagram of the Δ10, Δ10F and Δ10F4 minigene constructs. The 23-nt ISS has been expanded to show the nucleotide sequence. The GGGG motif and the poly-U tracts in Δ10F are boxed. Point mutations in Δ10F4 that disrupt the GGGG motif and the poly-U tracts are underlined. (D) K562 cells were nucleofected with either control or hnRNP C-specific siRNA (siC1). 24 hours later, these cells were nucleofected with either the Δ10F or the Δ10F4 minigene. After another 24 hours, real-time RT-PCR was performed on RNA from these cells to determine the ratio of exon 3- to exon 4-containing minigene products. Results are presented as an average of three biological replicates and the relative minigene E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells nucleofected with the same minigene, but without any siRNA. Error bars represent ± SEM.
Fig 2: SPMA Identifies ELAVL1 and TIA1 Motifs as Highly Enriched in Recurrent NSCLC Patients(A) Differential gene expression analysis was performed on samples from patients with untreated NSCLC tumors and patients with recurrent tumors.(B) Transite was used to identify RBPs whose targets were overrepresented among upregulated genes in the samples of recurrent tumors. Shown are 2 tables of k-mer-based TSMA and SPMA displaying RBPs with highly enriched motifs for TSMA and highly non-random motif enrichment patterns for SPMA. Among the top hits are ELAVL1, TIA1, and hnRNPC. P values were obtained by Monte Carlo sampling and corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure.(C) Spectrum plot from SPMA depicting the distribution of putative ELAVLI-binding sites across all of the transcripts. The transcripts are sorted by ascending signal-to-noise ratios. The transcripts downregulated in resistant samples relative to untreated samples are on the left, and those upregulated are on the right of the spectrum. The putative binding sites of ELAVL1 are highly enriched in transcripts upregulated in resistant cells (shown in red) and highly depleted in transcripts downregulated in resistant cells (shown in blue).(D) Spectrum plot of putative TIA1 binding sites using the same transcript order as in (C).(E) Enrichment of ELAVL1 targets in resistant NSCLC cells is recapitulated in an independent high-throughput sequencing of RNA isolated by CLIP (HITS-CLIP) experiment (publicly available data). The distribution of fold changes of transcripts that have ELAVL1-binding sites is shifted in the positive direction, even more so when the binding sites are in the 3' UTR. The p values were calculated with the 1-sided Kolmogorov-Smirnov test.(F) As in (E), transcripts with TIA1-binding sites are upregulated in resistant cells according to an iCLIP experiment, confirming results from SPMA.
Fig 3: Stimulation-induced repression of MKK7-E2 is consistent with position-dependent activity of CELF2. (A) Quantification (as in Fig. 3, n=3) of inclusion of exon 2 in the endogenous MKK7 gene in wildtype (WT) cells or those depleted for CELF2 by shRNA (KD, see Methods) cultured under unstimulated or stimulated conditions as in Fig. 5C. (B) Sequence of MKK7-E2 (gray) and immediate upstream (red) and downstream (orange) intronic sequence shown to be necessary and sufficient for stimulation-induced repression of MKK7-E2.18 Simplified schematics used in other panels shown at bottom. (C) UV crosslinking of RNAs from panel B with nuclear extract from unstimulated or stimulated Jurkat cells. Position of molecular weight standards are indicated, as well as CELF2, hnRNP C, HuR and SRSF3 as determined previously 18 and in panels D-E. (D) UV Crosslinking of RNAs indicated as in panel B with the Up and Down RNAs (panel B). –IP is total reaction, IP: GST, HuR and CELF2 are following immune-precipitation with antibodies specific for the indicated proteins. GST is used as a negative control for non-specific interaction, while HuR serves as a positive control for equal binding/IP. Position of CELF2 is indicated. (E) UV Crosslinking of RNAs indicated as in panel B with the Full RNA (panel B). –IP is total reaction, IP: HuR, CELF2, hnRNP C and U2AF65 are following immune-precipitation with antibodies specific for the indicated proteins. Position of CELF2 is indicated.
Fig 4: Coordinated expression of CELF2 and hnRNP C fine-tune splicing outcomes of target genes. (A) Schematic and summary of the consequence of expression of shRNA against CELF2 (shC2) or hnRNP C (shC) on the expression of both proteins, as listed on the top of the columns. ++, + and - indicate high, medium and low expression. (B) Left: Potential models of action of CELF2 and hnRNP C on splicing targets that account for both the coordinated regulation observed between these proteins and the subset of target genes that are impacted by depletion of only one or the other protein. See main text for detailed description of models. Right: Quantification of RT-PCR analysis of several target genes upon partial rescue of CELF2 or hnRNP C expression in the knock-down of the other gene. Values are derived from at least three biologically independent experiments. Error bars represent standard deviation. (C) Western blot of CELF2 and hnRNP C expression in cells used for RT-PCR analysis in panel B. +C and +C2 indicate the overexpression of hnRNP C or CELF2, respectively, from Flag-tagged cDNA vectors. The mobility of the cDNA-expressed proteins is different from the endogenous due to the Flag tag. HnRNP L is used as a loading control.
Fig 5: Decreased protein levels of splicing factors in striatum of Huntington’s disease mouse model. (A) Protein levels in striatum of wild-type and R6/1 mice and quantifications normalized with ß-actin (n = 7-20) (Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001). Data represent mean ± SEM. (B) Messenger RNA levels of Rbfox1 and Hnrnpc in three wild-type versus three R6/1 samples, according to the RNA-seq Salmon DEG-analysis. (C) HNRNPC and TIA1 immunohistochemistry staining in striatum of R6/1 and wild-type mice. (D) Double immunofluorescence with TIA1 (green) and HTT (EM48, red) antibodies in striatum of R6/1 mice. Empty arrows show TIA1 punctate pattern in the absence of mutant HTT (mHtt) inclusions. Filled arrows show discrete co-localization of TIA1 and mHtt fluorescence in inclusion bodies. Nuclei were counterstained with DAPI (blue). (E) Protein levels in striatum of wild-type and zQ175 mice and quantifications normalized with ß-actin (n = 6). (Student’s t-test; *P < 0.05). Data represent mean ± SEM.
Supplier Page from Abcam for Anti-hnRNP C1 + C2/HNRNPC antibody [4F4]