PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1993 January; 13(1): 487–495.
PMCID: PMC358928

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Abstract

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Andersen LB, Tommerup N, Koch J. Formation of a minichromosome by excision of the proximal region of 17q in a patient with von Recklinghausen neurofibromatosis. Cytogenet Cell Genet. 1990;53(4):206–210. [PubMed]
  • Baldari C, Cesareni G. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene. 1985;35(1-2):27–32. [PubMed]
  • Ballester R, Marchuk D, Boguski M, Saulino A, Letcher R, Wigler M, Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. [PubMed]
  • Ballester R, Michaeli T, Ferguson K, Xu HP, McCormick F, Wigler M. Genetic analysis of mammalian GAP expressed in yeast. Cell. 1989 Nov 17;59(4):681–686. [PubMed]
  • Banroques J, Delahodde A, Jacq C. A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing. Cell. 1986 Sep 12;46(6):837–844. [PubMed]
  • Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. [PubMed]
  • Berger SL, Birkenmeier CS. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. [PubMed]
  • Cawthon RM, Weiss R, Xu GF, Viskochil D, Culver M, Stevens J, Robertson M, Dunn D, Gesteland R, O'Connell P, et al. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell. 1990 Jul 13;62(1):193–201. [PubMed]
  • Colicelli J, Birchmeier C, Michaeli T, O'Neill K, Riggs M, Wigler M. Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc Natl Acad Sci U S A. 1989 May;86(10):3599–3603. [PubMed]
  • Dingwall C, Laskey RA. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. [PubMed]
  • Furth ME, Davis LJ, Fleurdelys B, Scolnick EM. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. [PMC free article] [PubMed]
  • Gibbs JB, Schaber MD, Marshall MS, Scolnick EM, Sigal IS. Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells. J Biol Chem. 1987 Aug 5;262(22):10426–10429. [PubMed]
  • Goldfarb DS. Nuclear transport. Curr Opin Cell Biol. 1989 Jun;1(3):441–446. [PubMed]
  • Gross M, Sweet RW, Sathe G, Yokoyama S, Fasano O, Goldfarb M, Wigler M, Rosenberg M. Purification and characterization of human H-ras proteins expressed in Escherichia coli. Mol Cell Biol. 1985 May;5(5):1015–1024. [PMC free article] [PubMed]
  • Gutmann DH, Wood DL, Collins FS. Identification of the neurofibromatosis type 1 gene product. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9658–9662. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Leff SE, Rosenfeld MG, Evans RM. Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem. 1986;55:1091–1117. [PubMed]
  • Li Y, Bollag G, Clark R, Stevens J, Conroy L, Fults D, Ward K, Friedman E, Samowitz W, Robertson M, et al. Somatic mutations in the neurofibromatosis 1 gene in human tumors. Cell. 1992 Apr 17;69(2):275–281. [PubMed]
  • Marchuk D, Drumm M, Saulino A, Collins FS. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. [PMC free article] [PubMed]
  • Marchuk DA, Saulino AM, Tavakkol R, Swaroop M, Wallace MR, Andersen LB, Mitchell AL, Gutmann DH, Boguski M, Collins FS. cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product. Genomics. 1991 Dec;11(4):931–940. [PubMed]
  • Martin GA, Viskochil D, Bollag G, McCabe PC, Crosier WJ, Haubruck H, Conroy L, Clark R, O'Connell P, Cawthon RM, et al. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. [PubMed]
  • Nikawa J, Cameron S, Toda T, Ferguson KM, Wigler M. Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. Genes Dev. 1987 Nov;1(9):931–937. [PubMed]
  • Nishi T, Lee PS, Oka K, Levin VA, Tanase S, Morino Y, Saya H. Differential expression of two types of the neurofibromatosis type 1 (NF1) gene transcripts related to neuronal differentiation. Oncogene. 1991 Sep;6(9):1555–1559. [PubMed]
  • Roberts B. Nuclear location signal-mediated protein transport. Biochim Biophys Acta. 1989 Aug 14;1008(3):263–280. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Sass P, Field J, Nikawa J, Toda T, Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. [PubMed]
  • Suzuki Y, Suzuki H, Kayama T, Yoshimoto T, Shibahara S. Brain tumors predominantly express the neurofibromatosis type 1 gene transcripts containing the 63 base insert in the region coding for GTPase activating protein-related domain. Biochem Biophys Res Commun. 1991 Dec 31;181(3):955–961. [PubMed]
  • Tanaka K, Matsumoto K, Toh-E A. IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):757–768. [PMC free article] [PubMed]
  • Tanaka K, Nakafuku M, Satoh T, Marshall MS, Gibbs JB, Matsumoto K, Kaziro Y, Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. [PubMed]
  • Tanaka K, Nakafuku M, Tamanoi F, Kaziro Y, Matsumoto K, Toh-e A. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol Cell Biol. 1990 Aug;10(8):4303–4313. [PMC free article] [PubMed]
  • Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. [PubMed]
  • Trahey M, McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. [PubMed]
  • Trahey M, Wong G, Halenbeck R, Rubinfeld B, Martin GA, Ladner M, Long CM, Crosier WJ, Watt K, Koths K, et al. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. [PubMed]
  • Tschumper G, Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. [PubMed]
  • Upadhyaya M, Cheryson A, Broadhead W, Fryer A, Shaw DJ, Huson S, Wallace MR, Andersen LB, Marchuk DA, Viskochil D, et al. A 90 kb DNA deletion associated with neurofibromatosis type 1. J Med Genet. 1990 Dec;27(12):738–741. [PMC free article] [PubMed]
  • Viskochil D, Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK, Culver M, Carey JC, Copeland NG, Jenkins NA, et al. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell. 1990 Jul 13;62(1):187–192. [PubMed]
  • Wallace MR, Andersen LB, Saulino AM, Gregory PE, Glover TW, Collins FS. A de novo Alu insertion results in neurofibromatosis type 1. Nature. 1991 Oct 31;353(6347):864–866. [PubMed]
  • Wallace MR, Marchuk DA, Andersen LB, Letcher R, Odeh HM, Saulino AM, Fountain JW, Brereton A, Nicholson J, Mitchell AL, et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science. 1990 Jul 13;249(4965):181–186. [PubMed]
  • Wang Y, Boguski M, Riggs M, Rodgers L, Wigler M. sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1. Cell Regul. 1991 Jun;2(6):453–465. [PMC free article] [PubMed]
  • Xu GF, Lin B, Tanaka K, Dunn D, Wood D, Gesteland R, White R, Weiss R, Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. [PubMed]
  • Xu GF, O'Connell P, Viskochil D, Cawthon R, Robertson M, Culver M, Dunn D, Stevens J, Gesteland R, White R, et al. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)