Neurofibromin (NF-1) is a protein that is encoded in humans, in the NF1 gene. NF1 is located on chromosome 17. Neurofibromin, a GTPase-activating protein that negatively regulates RAS/MAPK pathway activity by accelerating the hydrolysis of Ras-bound GTP. NF1 has a high mutation rate and mutations can alter cellular growth control, and neural development, resulting in various presentations (depending on the specific allele expressed) such as Watson syndrome and neurofibromatosis type 1 (NF1, also known as von Recklinghausen syndrome). Symptoms of NF1 include disfiguring cutaneous neurofibromas (CNF), café au lait pigment spots, plexiform neurofibromas (PN), skeletal defects, optic nerve gliomas, life-threatening malignant peripheral nerve sheath tumors (MPNST), pheochromocytoma, attention deficits, learning deficits and other cognitive disabilities.
Gene NF1 was cloned in 1990 and its product neurofibromin was identified in 1992. Neurofibromin, a GTPase-activating protein, primarily regulates the protein Ras. NF1 is located on the long arm of chromosome 17, position q11.2 NF1 spans over 350-kb of genomic DNA and contains 62 exons. 58 of these exons are constitutive and 4 exhibit alternative splicing ( 9a, 10a-2, 23a, and 28a). The genomic sequence starts 4,951-bp upstream of the transcription start site and 5,334-bp upstream of the translation initiation codon, with the length of the 5' UTR being 484-bp long. There are three genes that are present within intron 27b of NF1. These genes are EVI2B, EVI2A and OMG, which are encoded on the opposite strand and are transcribed in the opposite direction of NF1. EVI2A and EVI2B are human homologs of the Evi-2A and Evi-2B genes in mice that encode proteins related to leukemia in mice. OMG is a membrane glycoprotein that is expressed in the human central nervous system during myelination of nerve cells.
Promoter Early studies of the NF1 promoter found that there is great homology between the human and mouse NF1 promoters. The major transcription start site has been confirmed, as well as two minor transcription start sites in both the human and mouse gene. The major transcription start is 484-bp upstream of the translation initiation site. The open reading frame is 8,520-bp long and begins at the translation initiation site. NF1 exon 1 is 544-bp long, contains the 5' UTR and encodes the first 20 amino acids of neurofibromin. The NF1 promoter lies within a CpG island that is 472-bp long, consisting of 43 CpG dinucleotides, and extends into the start of exon 1. This CpG Island begins 731-bp upstream of the promoter and no core promoter element, such as a TATA or CCATT box, has been found within it. Although no core promoter element has been found, consensus binding sequences have been identified in the 5' UTR for several transcription factors such as Sp1 and AP2. A methylation map of five regions of the promoter in both mouse and human was published in 1999. This map showed that three of the regions (at approximately – 1000, – 3000, and – 4000) were frequently methylated, but the cytosines near the transcription start site were unmethylated. Methylation has been shown to functionally impact Sp1 sites as well as a CREB binding site. It has been shown that the CREB site must be intact for normal promoter activity to occur and methylation at the Sp1 sites may affect promoter activity. Proximal NF1 promoter/5' UTR methylation has been analyzed in tissues from NF1 patients, with the idea that reduced transcription as a result of methylation could be a "second hit" mechanism equivalent to a somatic mutation. There are some sites that have been detected to be methylated at a higher frequency in tumor tissues than normal tissues. These sites are mostly within the proximal promoter; however, some are in the 5' UTR as well and there is a lot of interindividual variability in the cytosine methylation in these regions.
3' UTR A study in 1993 compared the mouse NF1 cDNA to the human transcript and found that both the untranslated regions and coding regions were highly conserved. It was verified that there are two NF1 polyadenylated transcripts that differ in size because of the length of the 3' UTR, which is consistent with what has been found in the mouse gene. A study conducted in 2000 examined whether the involvement of the 3' UTR in post-transcriptional gene regulation had an effect on the variation of NF1 transcript quantity both spatially and temporally. Five regions of the 3' UTR that appear to bind proteins were found, one of which is HuR, a tumor antigen. HuR binds to AU-rich elements which are scattered throughout the 3' UTR and are thought to be negative regulators of transcript stability. This supports the idea that post-transcriptional mechanisms may influence the levels of NF1 transcript.
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