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 23a⌬/23a⌬ 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 23a⌬/23a⌬ 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.

A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor

“…Exon 23 encodes part of the GRD, which is the RAS regulatory domain of neurofibromin. An exon 23 splice variant inserts an alternative exon 23a, which decreases neurofibromin RASGAP activity 68 . In addition to this central GRD, neurofibromin contains other functional domains, most of which are of uncertain importance in the tumour suppressor function of neurofibromin (FIG.…”

Understanding the Biological Activities of Vitamin D in Type 1 Neurofibromatosis: New Insights into Disease Pathogenesis and Therapeutic Design

“…Finally, if exon 10a-2 is encoded, the produced isoform presents a transmembrane domain. This isoform is expressed in several human tissues, therefore it likely performs a housekeeping function in the intracellular membranes [ 271 , 299 , 300 , 301 , 302 ].…”

Cutaneous neurofibromas in the genomics era: current understanding and open questions

“…NF1 function can also be impaired by post-transcriptional alterations such as alternative splicing, microRNA-mediated repression of the transcript or excessive proteasomal degradation of the protein. 48 – 50 Consequently, detection of NF1 loss-of-function events and clarification of the NF1 genotype–phenotype relationship are existing challenges to the field. 51 Some studies have started to characterise the genotype–phenotype relationship between NF1 and cNFs, and these further suggest that there are a wide range of somatic NF1 mutations associated with cNF.…”