Regulation of Neuron-Specific Alternative Splicing of Neurofibromatosis Type 1 Pre-mRNA

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5′ splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3′ splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing.

ACKNOWLEDGMENTS

We thank the following individuals for providing antibodies and plasmids: Henry Furneaux at the University of Connecticut Health Center (GST-hHuD), Imed-Eddine Gallouzi at McGill University (GST-HuR), Alison Hall at Case Western Reserve University and Andrew Russo at the University of Iowa (CA77 cell line), Jerome Posner at the Sloan Kettering Cancer Center (Hu patient sera), and Ann-Bin Shyu at the University of Texas-Houston Medical School (HuR cDNA). Greg Matera's laboratory provided help with the siRNA knockdown experiments. We thank Helen Salz and Jo Ann Wise for critical reading of the manuscript.

This work was supported by an NSF grant (MCB-0237685) and an NIH grant (NS-049103-01) to Hua Lou. Hui Zhu was supported by a predoctoral fellowship from the American Heart Association (0415086B). Melissa Hinman is supported by a genetics training grant from NIH (T32GM008613).