Human Molecular Genetics, 2002, Vol. 11, No. 17 2037-2049
© 2002 Oxford University Press
hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-ß1
Institute of Human Genetics, University of Bonn, Bonn, Germany
Received May 7, 2002; Accepted June 18, 2002
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-ß1, 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-ß1 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-ß1 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.
* To whom correspondence should be addressed at: University of Bonn, Institute of Human Genetics, Wilhelmstr. 31, 53111 Bonn, Germany. Tel: +49 2282872344; Fax: +49 2282872380; Email: bwirth{at}uni-bonn.de
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