Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F

Abstract

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5' splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.

Figure 1. Genome wide analysis of alternative spliced events (ASEs) regulated by hnRNPH/F.

Oli-neu cells were treated with an siRNA, siF/H that targets both hnRNPH and F . 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 (www.genosplice.com). A. 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. 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. C. 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. D. 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. E. 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.

Figure 2. Changes in exon inclusion induced by silencing hnRNPH and F individually vs. both simultaneously.

Representative RT-PCR (n = 2) of the products derived from 5′ASEs in Oli-neu cells treated with siRNAs that target hnRNPH (siH), hnRNPF (siF) or both (siF/H). We selected ASEs that were shown to have a statistically significant change in exon inclusion by RT-PCR. Mock are control untreated Oli-neu cells. Each ASE is labeled with the gene ID number and the ae is shown (also refer to Figure 1E). The number shown below each lane represents the fold change in exon inclusion compared to the mock treated cells set at the value of 1. The PLP/DM20 splicing event shows the synergistic effect of hnRNPH/F knock down.

Figure 3. Frequency difference (FD) plot of G tracts in the intron downstream of internal cassette exons. A.

FD plot of intronic G triplets for decreased inclusion (down regulated) and increased inclusion (up regulated) 5′ splice sites (ss). FD is defined as the difference between the observed frequency of GGG in introns, calculated in a 30-nt window, and the mean frequency of GGG in 10 random permutations of the sequence in the same window, with an offset of 3nt between successive windows, as described . Black bars show the standard errors. B. FD plot of intronic G quadruplets and longer G tracts for down and up regulated 5′ ss. C. FD plot of G triplets in the intron downstream of intermediate and strong 5′ ss of exons with decreased inclusion (hnRNPH/F-activated). D. FD plot of G triplets in the intron downstream of intermediate and strong 5′ ss of exons with increased inclusion (hnRNPH/F-repressed).

Figure 4. Genes are differentially regulated at the transcriptional levels.

A. Representation of significant pathways whose genes are affected by knock down of hnRNPH/F. Thirty one Kegg pathways were significantly affected. Genes are either up- or down-regulated. B. Changes in gene expression were verified by Real Time RT-PCR in Oli-neu cells depleted of hnRNPH and F. Bar graphs represent the mean±SD of transcript levels of the indicated genes quantitated by Real Time RT-PCR in mock siRNA treated (Mock) and siF/H treated Oli-neu cells (n = 3). Oli-neu cells were treated with siF/H and harvested after 72 hrs in culture for Real Time RT-PCR analysis. The data are expressed as percent change of the treated vs. mock cells, the latter is set at the value of 1. ns = non significant, *p = 0.05. In parenthesis next to the gene name is shown the fold change in the microarrays.

Figure 5. Expression profile of the validated genes in developing oligodendrocytes in vivo.

CNPase-EGFP+ oligodendrocytes were isolated at post-natal day (P) 1, 10 and 21 and subjected to RT-PCR, Western blot analysis and Real Time RT-PCR. A. Representative RT-PCR of PLP/DM20 ratio and Western blot analysis of hnRNPH and F in developing oligodendrocytes. CNPase is a marker of differentiation and increases in P10 and P21 OL vs. P1 oligodendrocyte progenitor cells. β-tubulin is the loading control. B. Bar graphs represent the mean ± SD of transcript levels of the indicated genes quantitated by Real Time RT-PCR in EGFP+ oligodendrocytes in vivo (n = 3). The fold change detected in the microarray is shown in parenthesis next to the gene name. ns = non statistically significant. **≤0.01.