PLP1 alternative splicing in differentiating oligodendrocytes: characterization of an exonic splicing enhancer.
Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5' donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406-412) reduced PLP 5' donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382-389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406-412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5'splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration-dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5' donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes.
Wang
Erming
E
Department of Neurology, University of Kentucky, Lexington, Kentucky 40536-0284, USA.
Huang
Zhong
Z
Hobson
Grace M
GM
Dimova
Neviana
N
Sperle
Karen
K
McCullough
Andrew
A
Cambi
Franca
F
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D019070:Cell Lineage / Q000502:physiology; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D005091:Exons; D006801:Humans; D009154:Mutation; D018991:Myelin Proteolipid Protein / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009836:Oligodendroglia / Q000502:physiology*; D011401:Promoter Regions, Genetic; D022821:RNA Splice Sites; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D018991:Myelin Proteolipid Protein; D009419:Nerve Tissue Proteins; C489071:Plp1 protein, rat; D022821:RNA Splice Sites