Genome wide analysis of alternative spliced events (ASEs) regulated by hnRNPH/F.

<p>Oli-neu cells were treated with an siRNA, siF/H that targets both hnRNPH and F <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051266#pone.0051266-Wang3" target="_blank">[12]</a>. RNA was used to generate probes to hybridize with the Affymetrix Mouse exon 1.0ST array featuring ∼ 1 million exon clusters and 1.4 million probe sets. We have analyzed splicing and transcript levels using the EASANA® from GenoSplice technology (<a href="http://www.genosplice.com" target="_blank">www.genosplice.com</a>). <b>A.</b> RT-PCR amplification of the endogenous PLP and DM20 transcripts. Schematic of the PCR products is shown. siF/H treatment induces a two-fold increase in the inclusion of exon 3B. Percent inclusion of exon 3B is shown. <b>B.</b> Western blot analysis of hnRNPH and F expression. More than 70% reduction of hnRNPH/F is induced by the siF/H treatment. hnRNPA1 is used as loading control. <b>C.</b> Pie chart showing the ASEs regulated by hnRNPH/F. Splicing of 252 exons was differentially regulated by knock down of hnRNPH/F. The types of spliced events are shown. <b>D.</b> Pie charts show the relative abundance of included (hnRNPH/F-repressed) and excluded (hnRNPH/F-activated) exons. For four of the six categories of alternative spliced events a greater number of exons are excluded (i.e. hnRNPH/F-activated) by depletion of hnRNPH/F. <b>E.</b> RT-PCR of alternative 5′ splice sites. Representative RT-PCR of 5′ASEs that were examined by semiquantitative RT-PCR analysis in siF/H treated vs. untreated Oli-neu cells (mock) (n = 3). Each ASE is labeled with the gene ID and the alternative spliced exon (ae) is shown. The ASEs that were validated by RT-PCR are shown in bold. The others, not bolded, demonstrated a change that was in the opposite direction of that detected in the arrays. Bar graphs represent the percent change of the exon inclusion ± SD in the siF/H treated cells vs. untreated cells set at 1 (n = 3). *≤0.05 and **≤0.01.