Neurofibromatosis Type 1 Alternative Splicing Is a Key Regulator of Ras Signaling in Neurons

Abstract

Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling and may be important in modulating Ras-related neuronal functions.

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Neurofibromatosis Type 1 Alternative Splicing Is a Key Regulator of Ras Signaling in Neurons

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (grants NS049103 to H.L., F31 NS647242 to M.N.H., T32 GM08613 to M.N.H., and S10RR021228 and S10RR024536 [National Center for Research Resources shared-instrumentation grants for the Leica DM6000 wide-field microscope and the GE Healthcare Typhoon Trio variable-mode imager]) and the U.S. Department of Defense (grant NF060083 to H.L.).

We thank David LePage and the Case Transgenic and Targeting Facility for help with gene targeting, David Friel for assistance with neuron experiments, and Nancy Ratner for providing the anti-Nf1 antibody and Schwann cell RNA. We also thank Jo Ann Wise for a critical reading of the manuscript and Helen Salz, Ronald Conlon, Evan Deneris, and members of the Lou laboratory for helpful discussions.