hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1.
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.
Mesh Terms:
Alternative Splicing, Animals, Cell Line, Cyclic AMP Response Element-Binding Protein, Enhancer Elements, Genetic, Exons, Heterogeneous-Nuclear Ribonucleoproteins, Humans, Mice, Motor Neurons, Muscular Atrophy, Spinal, Nerve Tissue Proteins, Nuclear Proteins, Precipitin Tests, RNA, Messenger, RNA-Binding Proteins, Recombinant Proteins, SMN Complex Proteins, Survival of Motor Neuron 1 Protein, Survival of Motor Neuron 2 Protein, Transcription, Genetic, Y Chromosome
Alternative Splicing, Animals, Cell Line, Cyclic AMP Response Element-Binding Protein, Enhancer Elements, Genetic, Exons, Heterogeneous-Nuclear Ribonucleoproteins, Humans, Mice, Motor Neurons, Muscular Atrophy, Spinal, Nerve Tissue Proteins, Nuclear Proteins, Precipitin Tests, RNA, Messenger, RNA-Binding Proteins, Recombinant Proteins, SMN Complex Proteins, Survival of Motor Neuron 1 Protein, Survival of Motor Neuron 2 Protein, Transcription, Genetic, Y Chromosome
Hum. Mol. Genet.
Date: Aug. 15, 2002
PubMed ID: 12165565
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