title: hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1.
abstract: Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-beta1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein-protein interaction of hnRNP-G with Htra2-beta1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-beta1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far.
authors.affiliation: Institute of Human Genetics, University of Bonn, Bonn, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Hofmann
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Wirth
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein; D004742:Enhancer Elements, Genetic; D005091:Exons / Q000235:genetics*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009046:Motor Neurons; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009687:Nuclear Proteins; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D055532:SMN Complex Proteins; D000068103:Serine-Arginine Splicing Factors; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014158:Transcription, Genetic; D014998:Y Chromosome
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C501499:RBMY1A1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C501500:Rbmy protein, mouse; D011994:Recombinant Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; C526472:Smn1 protein, mouse; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; C097083:TRA2B protein, human; D000068103:Serine-Arginine Splicing Factors