Neurofibromatosis type 1 (NF1), a common human genetic disease affecting the nervous system, is characterized by phenotypes ranging from increased tumor susceptibility to learning disabilities. The NF1 gene inactivates the oncogene Ras through its GTPase activating protein (GAP) domain. NF1 alternative exon 23a is located within the GAP domain and is regulated by a number of factors, including the Hu family of RNA-binding proteins, to be skipped in brain but included in other tissues.
To study the molecular and biological functions of this regulated splicing event, we engineered mouse embryonic stem (ES) cells either with endogenous Nf1 exon 23a deleted (Nf1 23aΔ/23aΔ cells) or with the splicing signals surrounding Nf1 exon 23a mutated to more closely match consensus sequences (Nf1 23aIN/23aIN cells) such that the exon is constitutively included. Both as ES cells and as ES cell-derived neurons, Nf1 23aIN/23aIN mutants have higher levels of active Ras than Nf1 23aΔ/23aΔ cells. In addition, Nf1 23aIN/23aIN neurons have elevated levels of phosphorylated ERK1/2 downstream of Ras. We generated Nf1 23aIN/23aIN mice with constitutive Nf1 exon 23a inclusion in all tissues, and found increased Ras activation in their brains. Taken together, these results strongly suggest that the regulated inclusion of Nf1 exon 23a modulates Ras signaling both in cells and in mice. Nf1 23aIN/23aIN mice are viable and will serve as useful tools for studying the roles of Nf1 alternative splicing in processes such as learning and tumor suppression.
Given the functional importance of NF1 exon 23a, we also studied the mechanisms by which its inclusion is regulated by HuC, an Hu protein family member. Specifically, we used deletion mutants to study how the domains of HuC, a poorly characterized splicing regulator, contribute to the regulation of NF1 exon 23a and two other alternative exons. We found that the HuC N-terminal region is dispensable for splicing regulation, the three RNA recognition motifs (RRMs) are required for splicing regulation, and the hinge region is required for optimal regulation of only some splicing targets. RRM1 and RRM2 recognize and bind to RNA targets, while the hinge region and RRM3 contribute to HuC-HuC self interaction.