Complex splicing pattern generates great diversity in human NF1 transcripts

Abstract

Background: Mutation analysis of the neurofibromatosis type 1 (NF1) gene has shown that about 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts. Some of these transcripts were also found in fresh lymphocytes of healthy individuals, albeit typically at a very low level. Starting from this initial observation, we were interested to gain further insight into the complex nature of NF1 mRNA processing.

Results: We have used a RT-PCR plasmid library based method to identify novel NF1 splice variants. Several transcripts were observed with specific insertions/deletions and a survey was made. This large group of variants detected in one single gene allows to perform a comparative analysis of the factors involved in splice regulation. Exons that are prone to skipping were systematically analysed for 5' and 3' splice site strength, branch point strength and secondary structure.

Conclusion: Our study revealed a complex splicing pattern, generating a great diversity in NF1 transcripts. We found that, on average, exons that are spliced out in part of the mRNA have significantly weaker acceptor sites. Some variants identified in this study could have distinct roles and might expand our knowledge of neurofibromin.

Figure 1

Representative restriction pattern of eight colonies of NF1-F4 digested with AvaII. (1)-(4), (7), (8): full length NF1-F4, (5): NF1-ΔE30, (6): NF1-Δ[E30, E36/E37]

Figure 2

Schematic representation of all splice variants detected in five overlapping NF1 fragments. In frame events are indicated in green whereas out of frame events are red. Transcript variants described in blue are identical to misspliced transcripts, described previously for NF1 patients, caused by a genomic mutation. The number of colonies detected containing a specific variant is indicated between brackets.

Figure 3

Simulations of secondary structure. Secondary structure of the sequence surrounding the donor site of exon 7 (A, B), exon 4b (C, D) and exon 8 (E-F). A and C: lowest free-energy structures; B and D: first higher energy-structure; E and F: prediction using a window of (-50, +50) (E) or (-56, +44) (F) with respect to the splice site.