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.
Highlights
HnRNPH and F control alternatively spliced events (ASE) by binding to G tracts positioned in close proximity to the 59 or 39 splice sites, with G triplets being the basic recognition motif [1,2,3,4,5]. hnRNPH and F can either enhance or inhibit the alternatively spliced exon and the magnitude of the effect is dependent on the length of the G tracts, the intronic vs. exonic position and the strength of the 59 ss [6,7,8,9,10]
We have shown that hnRNPH/F regulate the major myelin proteolipid protein (PLP)/DM20 ratio predominantly by enhancing the selection of the DM20 59 splice site through long G tracts positioned in exon 3B immediately downstream of the DM20 59ss [11,12,13]
Treatment of Oli-neu cells with 80 nM siF/H reduces hnRNPH/F expression greater than 70% (Fig. 1B and [11]), which results in a two-fold increase in the PLP/DM20 ratio derived from the endogenous PLP transcript (Fig. 1A, [11])
Summary
HnRNPH and F control alternatively spliced events (ASE) by binding to G tracts positioned in close proximity to the 59 or 39 splice sites (ss), with G triplets being the basic recognition motif [1,2,3,4,5]. hnRNPH and F can either enhance or inhibit the alternatively spliced exon and the magnitude of the effect is dependent on the length of the G tracts, the intronic vs. exonic position and the strength of the 59 ss [6,7,8,9,10]. We have shown that hnRNPH/F regulate the major myelin proteolipid protein (PLP)/DM20 ratio predominantly by enhancing the selection of the DM20 59 splice site through long G tracts positioned in exon 3B immediately downstream of the DM20 59ss [11,12,13]. Unlike other ASEs, hnRNPH and F exert a novel synergistic regulation of the PLP alternatively spliced event and their function is not redundant [12]. The alternative splicing of PLP is a differentiation dependent event in the oligodendrocytes (OL), the myelin producing cells of the central nervous system (CNS). Endogenous hnRNPH and F expression is high in oligodendrocyte progenitor cells (OPC) and decreases in differentiated OL in vitro at the time when the PLP/ DM20 ratio increases [12]. The down regulation of hnRNPH/F is temporally related to the transition of oligodendrocyte progenitor cells to differentiated OL, suggesting that hnRNPH/F may contribute broadly to differentiationinduced changes in gene splicing and expression that occur as part of the OL differentiation program
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