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16, 1992
MOLECULAR
CLONING
NEUROFIBROMATOSIS DOMAIN
RELATED
TYPE
OF A cDNA CODING
1 PROTEIN
ISOFORM
TO ras GTPase-ACTIVATING
984-990
FOR LACKING
THE
PROTEIN’
Hiroyuki Suzuki, Kazuhiro Takahashi,Yasuhiko Kubota and Shigeki Shibahara* Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980, Japan Received
August
1, 1992
Summary Neurofibromatosis type 1 (NFl) is an autosomal dominant neurocutancous disorder, and a genelinked to NFl was recently identified. Its gene product (NFl protein) contains a domain functionally related to mammalianras GTPase-activating protein (GAP). Here, we cloned a cDNA coding for NFl protein isoform lacking the region related to GAP from a oligo(dT)-primed cDNA library of humanplacenta. This cDNA carries the insert of about 2.4 kb, coding for a protein of 551 amino acid residues,which sharesthe sameamin@ terminal 547 residueswith authentic NFl protein. We show that NFl mRNAs of about 2.9, 11, and 13 kb are expressedin human tissues,and that the isolated cDNA may represent the 0 1992ncademlc Press,1°C. 2.9-kb transcript.
Neurofibromatosistype 1 (NFl) or von Recklinghausen’sdiseaseis an autosomaldominant disorder,affecting oneper 3000-4000individuals, and is characterized by the presenceof cafk au lad skin spots,multiple neurofibromas, and Lisch nodule (1,2). Recently, a gene linked to NFl was identified by positional cloning (3-Q and structural analysis of its gene transcript revealed an open reading frame of 2818 amino acids (6). A portion of NFl protein sequence showssimilarity to mammalianras GTPase-activating protein (GAP) and yeast IRA proteins (7,8), which are known to down-regulatemammalianrasand yeast RAS proteins, respectively (9,lO). The GAP-related domainof NFl protein wasthen shownto possessGAP activity (ll13). Furthermore,NFl protein was shownto be involved in the negative control of rasin intact cells and in the inhibition of cell proliferation (14), suggestingthat the NFl gene is a tumorsuppressorgene (14-16). NFl gene product was also termed neurofibromin (17), since it is abundantin the nervous system. Recently, we and other group reported the presenceof two types of NFl mRNAs probably produced by alternative splicing; type I is authentic NFl mRNA and type II has the 63-bp isequence data from this article have beendepositedwith the EMBWGenBank Data Libraries underAccessionNo. D 12625. *To whom correspondenceshouldbe addressed. Abbreviations: NFl, Neurofibromatosis type 1; GAP, ras GTPase-activating protein; kb, kilobases;PCR, polymerasechain reaction; bp, basepairs. 0006-29 1X/92 $4.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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insertion in the GAP-related domain (1819).
Interestingly, expression of these two types of mRNAs may change during cell growth status (18), although the exact function of authentic NFl protein remains to be elucidated.
To establish the function of NFl gene product, we
attempted to clone a full-length NFl cDNA from human cDNA libraries. We thus isolated a cDNA for NFl protein isoform lacking the GAP-related domain. Its deduced protein, consisting of 551 amino acid residues, shares the same amino-terminal authentic NFl protein, except for four residues at its carboxy-terminus.
547 residues with
We then confirmed the
expression of the NFl gene transcripts coding for this NFl protein isoform in human tissues. Furthermore, we cloned a partial cDNA coding for the 3’-portion of authentic NFl mRNA, providing the first evidence that authentic NFl mRNA contains the 3’-untranslated about 3 kb.
Materials
region of
and Methods
Preparation ofRNA - Total RNA was prepared from human kidney, placenta and HeIa cells by guanidium thiocyanate-CsCl method (20). Poly(A)+ RNA was isolated by oligo (dT)cellulose chromatography (21). cDNA cloning for NFl protein - A cDNA library was constructed from 4 pg of poly(A)+ RNA of human placenta by the method of Okayama and Berg (22,23). A cDNA library of a human basophilic leukemia cell line (KU-812-F) was similarly constructed as described (24). Total plasmid DNA (1Opg) of each cDNA library was linearized with SalI, electrophoresed on agarose gels, transferred to a nylon membrane filter (Zeta-probe membrane, Bio-Rad), and hybridized with each of sap-labeled cDNA fragments generated by polymerase chain reaction (PCR). The DNA probes used were amplified from kidney cDNAs using synthetic PCR primers designed to cover the regions coding for the amino-terminus and carboxy-terminus of NFl protein (7). Synthesized were two sense primers, AA’ITGGGAAGATAACTCTGT (nucleotide numbers 1003/1022) and GTAGCCACAGGTCCGGCTCTC (6271/6290), and two anti-sense primers TGAGGGCTTATACGAAAGCA (complementary to 1147/1166) and TITAAGCAACTCTCAAGTGC (complementary to 6760/6779). The nucleotide residues were renumbered from the A residue of the initiating ATG codon (6). The amplified cDNA fragments were labeled with [a-32P]dCTP (3000 Ci/mmol, Amersham) by the method of Feinberg and Vogelstein (25). According to the size of hybridizable bands detected, the target cDNA fragments were purified, self-ligated, and used to transform E. coli DHScr. Screening such size-fractionated cDNA libraries enriched with the target clones, two different clones were isolated, and designated pHN-3 and pHN-2, representing the cDNA clones derived from placenta and a leukemia cell line, respectively. Nucleotide sequence was determined by the dideoxy chain-termination method (26) using Sequenase (USB) and BcaBEST sequencing kit (Takara). Northern blot and Sl nuclease-mapping analyses - Poly(A)+ RNA, denatured with glyoxal/dimethyl sulfoxide, was electrophoresed on an 1.0% agarose gel in 10 mM sodiumphosphate buffer (pH 7.0) (27) transferred to a nylon membrane filter (Zeta-probe membrane, Bio-Rad), and fixed with UV-linker (Stratalinker 1800, Stratagene). The HincII/PstI fragment (196/l 113) of pHN-3 was inserted into pBluescript KS vector (Stratagene), and a 32P-labeled riboprobe was prepared according to the method of Melton et al. (28). Hybridization was carried out in the mixture of 1.5x SSPE (0.225M NaCl, 15mM NaH2P04, 1.5mM EDTA, pH7.4) 50% formamide, 1% SDS, 0.5% skim milk, 0.5 mg/ml salmon testis DNA, and 0.2 mg/ml yeast tRNA. The hybridized filters were extensively washed at 50°C with 2x SSC, 0.1% SDS, and with 0.1x SSC, 0.1% SDS. The washed filters were then rinsed in 2x SSC, treated with RNase A (l&ml) in 2x SSC for 7 min at room temperature, washed in 0.1x SSC, 0.1% SDS at 50°C for 30 min, and then subjected to the detection with Bioimage Analyzer (Fuji BAS2000). Sl nuclease-mapping analysis was carried out as described previously (29). The Sl probe was prepared from a subclone carrying the ApaI/BamHI fragment of pHN-3 (1548/vector) in the SmaI site of pUC119. Both ends were converted to blunt-ends prior to 985
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ligation, and the BamHI site is located in the Okayama-Berg vector (23). Total RNA (20 pug) was hybridized with the HhaI/NcoI fragment (vector/1796) derived from this subclone,which was end-labeledat the NcoI site with [Y-3zPJATP (7000 Ci/mmol, ICN). Hybridized nucleic acids were digested with 20 units of Sl nuclease(Boehringer-Mannheim) and the protected fragmentswere analyzedon a 5% polyacrylamidegel containing 7 M urea. Radioactive signals were detectedby exposingthe dried gel to X-ray films (X-AR5, Kodak) at -85°C.
Results and Discussion Isolation aud characterization of NFl cDNA clones- To confirm the presenceof NFl cDNA clonesprior to screening,we analyzed total plasmidDNA derived from several human cDNA libraries. We thus detecteda band of about 5.5 kb with the 5’-probe in total plasmid DNA linearizedwith SalI, derived from a placentacDNA library, and detecteda 9-kb signal with 3’probe in leukemia cell cDNAs (data not shown). Since thesepositive fragments include the vector sequenceof about 3 kb (23) the actualsizesof thesecDNAs are smaller than that of the published NFl cDNA of more than 9 kb (6). We thus isolated two different cDNA clones, designated pHN-3 and pHN-2, from the size-fractionated cDNA libraries of placenta and leukemiacells, respectively (Fig.1). It shouldbe noted that thesecloneswere isolatedfrom the oligo(dT)-primed cDNA libraries and thus contain the 3’-untranslated region including a poly(A)-tail. H lkb
A NFl
mRNA
3
NF1 cDNA
I
pHN-3
,
PHN-2
I
Northern
probe
(v
I
zix c---B* Sl
probe
Figure 1. Schematic representation of NFl mRNA and its isolated cDNAs. (A) Relationship between NFl mRNA and isolated cDNAs. NFl mRNA is schematically drawn; box represents the protein-coding regionpublished (6), and solid lines, 5’ and 3’untranslated regions determined in this study. A slashed box indicates the GAP-related domain shown as GRD, and stippled boxes indicate the regions related to IRA. Cloned cDNAs for NFI mRNA are also drawn; closed boxes represent the protein-coding regions and solid lines, 5’ and 3’-untranslated regions. Two cDNAs cloned in this study are shown below the published sequence indicated as NFl cDNA. Note that the nucleotide sequence near the 3’-end of pHN-3 differs from the published sequence (6) (see Fig.2). The 5’-end of the insert cDNA carried by pHN-2 is located in the region near the nucleotide residue 5700 (indicated with two vertical lines). (B) Restriction map of pHN-3, showing only relevant or unique sites. The probes used in Fig.3 are also indicated. The Sl probe was the HhaI/NcoI fragment (vector/1796), endlabeled at the Ncol site (indicated by an asterisk); a dotted line represents the sequence of pUC119 and a solid line represents the protected fragment.
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ATCTCTAGCTCSCTCGCGCTCC TCTCCCCGGGCCGTGGAAAGGATCCCACTTCCGGTGGGGTGT~ATGG~GG~GT~T~GGA~TGTGATGG~TGTGGGGAGA~GG~G~TAGT
-361
CTTGCCAACGCCCCCTTTCCC
-181
-271
TCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACA~TGGGAGC~TG~A~T~~A~AGA~~~T~T~~TTG~~T~TT
-91
CCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGC~~ACCCTT~~~T~~G~CG~~~~~~GG~~G~GGGGAGGA~
-1
ATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAG~T~~~AATAAAAA~AGGA~AG~AGAA~A~A
90 30
MAAHRPVEWVOAVVSRFDEOLPI KTGOONT CATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACT
180 60 270
HTKVSTE HNKECLINI SKYKFSLVISGLTT ATTTTAAAGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTG I L K N VNNMRI FGEAAE K N L Y LSOLIIL GAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTT E K C LAGOPKD TMRLDETMLVKOL L P E CTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTC L
H
T
C
R
E
G
N
0
H
A
A
E
L
R
N
S
A
S
G
V
L
F
S
L
S
D
T
L
I
C
H
F
90 360 120
C
N
N
F
450 150
E
L
L
AATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTA STRLDELTVCSEDNVDVHDI NAVFSRI
540
CAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTA 0 Y I NVDCAKLKRLL K E T A F KFKALKKVAOL GCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACT~TACCAGATCCCACAGACT
210 720 240 610
AVINSLE KAFWNWVENYPDEFT KLYOIPOT GATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAA VDGFAESTKR KAAVWPLO D M A E C A E K L F D L
210 900 300
ATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTG IILLILCPEIIDDI SKDVVDENNMNKKLFL GACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCA RKALAGHGGS A C V K D S L R 0 L TESAAI
Q90 L
C
K
A
L
F
N
P
360
N
1 I10 390
AGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGC?TTTTAATCCA S
T
Y
I
N
W
E
D
N
S
V
I
FLLVOSMVVD
L
K
N
180 830
L
AGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAAC S K P F SRGSOPADVDLMI DCLVSCFRI
S
P
H
330 1060
AACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAAT NOHFKI CLAONSPSTFHYVLVNSLHRIITN TCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCA
1260 420 1360
S A L DWWPKIDA VYCHSVE LRNMFGETLHKA GTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGC~CCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAA R M A-P S L T F KEKVTSLKFKEK VOGCGAHPAI
450 I440 400
CCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCAT~GTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTTGTAAT SYKYLLLSM-VKLI HADPKLLLCN PTDLETR
1530 510
CCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAG TOGSTAELITG LVOLVPOSHMPE P RKOGPE
1620 540 1710
ATTGCTCAGGAAGCAATGGAGG~AAGGGGAAAATGAATTCCATGTTCTTGAAGGAAAGACTGTAACTATGTACATTCATGATGTTCCTTTI IAOEAME R G K t
551
GTGTGTGGTTTCTGTGAGTAACAGGTAGATGTCATTTCTGGAAATGGTATGTTTATGTCTATACATTGTTTTATAAAACTCCATGGAGA AGAAGGGGTTTACTTGCTTTGTATCACATAGCAATAACATTGTACAAATTCTGATGCTTAATAAAATAGTTCGAGATTTTC
1000 (A)n
1882
Figure 2. Nucleotide sequence of a cDNA coding for NFl protein isoform and its deduced amino acid seauence. The nucleotidc seauencc of DHN-3 is shown and is numbered in the 5’ to 3’ direct& from the A residue of the&initiating methionine codon ATG. The nucleotides on the 5’ side of residue 1 are indicated by negative numbers. The deduced amino acids are shown below the nucleotidc sequence and arc numbcrcd beginning with the initiating methionine. Boxed are the sequences newly identified. Double underlines indicate base changes at 702, 1389, and 1488. A putative poly-adcnylation signal is underlined, and a poly(A)-tail is shown as (A)n.
pHN-3 carriesthe cDNA insert of only about 2.4 kb, although pHN-3 was isolated by using the S-probe. The nucleotidesequenceof its cDNA insertwas then determined(Fig.2). pHN-3 contains the same5’-untranslatedregion asthe publishedNFl cDNA except that its length is longer by about 168 bp than that of the publishedNFl cDNA. The initiating methionine codon nucleotideresiduesl-3) was assignedaccording to the one of the authentic NFl cDNA reportedby Marchuk et al. (6). The nucleotidesequencefrom the initiation codon to the residue (ATG,
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1642 is identical to the sequence published previously, except for three bases as indicated in Fig.2. The nucleotide sequence downstream from position 1643 is different from that of NFl cDNA, and a codon for termination (TGA) is present at 1654/1656. Thus, pHN-3 contains the open reading frame of 1653 nucleotides, coding for 551 amino acid residues with a molecular weight of 62,300. The protein encoded by pHN-3 contains the four amino acid residues at its carboxyl terminus, which are not present in authentic NFl protein.
It is remarkable that this
isoform lacks the segment coding for GAP-related or IRA-related region (Fig.1). Since the nucleotide sequence AG/GTAAG (1640/1646) conforms to the consensus sequence of the donor site for splicing (30), it is conceivable that a part of the sequence downstream from 1642 may be spliced out as an intron for some NFl gene transcript. Three base changes were found in pHN-3 as described above and may represent polymorphism. residue 1488 leads to the amino acid substitution;
One base change at nucleotide
namely, pHN-3 contains the codon ATG
(1486-1488) for Met at 496, while the published cDNA contains ATA codon for Ile (7). This base change was also found in other NFl cDNA (6). Two base changes, leading to no amino acid substitutions, are found at nucleotide residues 702 and 1389; pHN-3 contains the A residue at both sites, while the published cDNA contains G and C residues, respectively (6,7). pHN-2 was characterized by restriction endonuclease mapping and a part of its insert DNA was sequenced. pHN-2 carries a cDNA of about 6 kb, coding for the carboxy-terminal portion of authentic NFl protein and the 3’-untranslated
region of about 3 kb. This size of the 3’-
untranslated region is similar to its assumed length of about 4 kb (6).
We thus propose that
authentic NFl mRNA contains the 3’-untranslated region of about 3 kb. Expression of the mRA4 codittg for NFl protein isoform - We investigated the expression of the transcripts coding for NFl protein isoform by Northern blot analysis. The cRNA probe used contains the region which is common to the transcripts coding for authentic NFl protein and its isoform (see Fig.1). We were thus able to detect at least three species of mRNAs about 13, 11, and 2.9 kb in both kidney and placenta poly(A)+ RNA (Fig.3A).
of
It seems likely
that the NFI mRNA of 13 or 1I kb may contain the 3’-untranslated region of 3 kb because the size of published cDNA sequence is more than 9 kb (6). This is the first report showing multiple species of NFl mRNAs detected by Northern blot analysis, be generated by alternative splicing. To confirm the expression represented by pHN-3, we designed Sl mapping analysis (Fig.3B).
These NFl mRNAs may of the 2.9-kb NFL mRNA The protected fragment of
expected size (249 bases) was detected in kidney, placenta and HeLa cell RNA, indicating that the 2.9-kb mRNA is indeed expressed. It remains to be elucidated whether the amino-terminal NFl protein isoform is actually expressed. To answer this question, we are currently producing antibodies against the amino-terminal domain of NFI protein. Implications for thepresence ofNF1 protein isoform - NFl protein is a molecule of 280 kDa (31), but its functional domain is determined only in the GAP/IRA-related region which is located in the center of the molecule. The function of other regions of NFl protein, including the amino-terminal domain, remains to be elucidated. In present paper, we suggest the existence of a NFl protein isoform possessing only amino-terminal portion of NFl protein and lacking GAP/IRA-related domain. Thus, it seemsunlikely that such an isoform could function as an 988
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Figure 3. Expression of the 2.9-kb mRNA coding for NFl protein isoform. (A) Northernblot analysis.Eachlanecontained 2.5p”gof poly(A)+RNA preparedfromhuman
kidney or placenta. A closedarrowheadindicatesthe transcriptsof about2.9 kb. A small horizontalbar indicatesthe origin of lanes. (B) Sl nuclcasemappinganalysis. Eachlane containedthe protectedproductswith yeasttRNA or total RNA preparedfrom the indicated sources.An openarrowheadindicatesthe Sl probeof 425bases,anda closedarrowhead indicatestheprotectedfragments, representing the1.9-kbmRNA. The sizemarkersusedwere theend-labeled 0X-174DNA digestedwith HacIII, andgivenin bases. upstreamregulator for ras protein. However, if the amino-terminal domain of NFl protein is involved in binding to somecellular protein, this NFl isoform may competewith NFl protein for binding to sucha protein, leadingto the altered expressionof GAP-like activity of authentic NFl protein. Alternatively, such an interaction involving this isoform may result in operating the ras signal transduction system in a manner that is independent of ras protein. These possibilitiesare consistentat leastin part with a model that NFl protein is a downstreameffector of ras protein (32,33). Elucidation of the function and regulation of this isoform may help to understandthe role of NFl protein in cellular signaltransductionsystemand the molecularbasis of the diverse clinical manifestationsof NFl. Acknowledgments This work was supportedin part by a Grant-in-Aid for Encouragementof Young Scientiststo H.S. from the Ministry of Education, Scienceand Culture of Japan,andby a grant to S.S. from the TakedaScienceFoundation. References
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6. Marchuk, D.A., Saulino, A.M., Tavakkol, R., Swaroop, M., Wallace, M.R., Anderson, L.B., Mitchell, A.L., Gutmann, D.H., Boguski, M. and Collins, F.S. (1991) Gerlomics 11, 931-940 7. Xu, G., O’Connell, P., Viskochil, D., Cawthon, R.M., Robertson,M., Culver, M., Dunn, D., Stevens, J., Gesteland, R., White, R., and Weiss, R. (1990) Cell 62, 599-608 8. Buchberg, A.M., Cleaveland, L.S., Jenkins, N.A. and Copeland, N.G. (1990) Nuture 347,291-294 9. Zhang, K., DeClue, J.E., Vass, W.C., Papageorge, A.G., McCormick, F. and Lowy, D.R. (1990) Nature 3 4 6, 754-756 10. Tanaka,K., Nakafuku, M., Satoh, T., Marshall, M.S., Gibbs, J.B., Matsumoto, K., Kaziro, Y. andToh-e, A. (1990) Cell 6 0, 803-807 11. Xu, G., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss, R. andTamanoi, F. (1990) Cell 63, 835-841 12. Martin, G.A., Viskochil, D., Bollag, G., McCabe, P.C., Crosler, W.J., Haubruck, H., Conroy, L., Clark, R., O’Connell, P., Cawthon, R.M., Innis, M.A., and McCormick, F. (1990) Cell 63, 843-849 13. Ballester, R., Marchuk, D., Boguski, M., Saulino, A., Letcher, R., Wigler, M., and Collins, F.S. (1990) Cell 63, 851-859 14. Basu, T.N., Gutmann, D.H., Fletcher, J.A., Glover, T.W., Collins, F.S. and Downward, J. (1992)Nature 356, 713-715 15. Li, Y., Bollag, G., Clark, R., Stevens, J., Conroy, L., Fults, D., Ward, K., Friedman, E., Somowits, W., Robertson, M., Bradley, P., McCormick, F., White, R. and Cowthon, R. (1992) Cell 69, 275-281 16. DeClue, J.E., Papageorge,A.G., Fletcher, J.A., Diehl, S.R., Ratner, N., Vass, W.C. and Lowy, D.R. (1992) Cell 6 9,265-273 17. Daston, M.M., Scrable, H., Nordlund, M., Sturbaum, A.K., Nissen, L.M. and Ratner, N. (1992) Neurolz 8,415-428 18. Suzuki, Y., Suzuki, H., Kayama, T., Yoshimoto, T., and Shibahara, S. (1991)BiocAem. Biophvs.
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Pages
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MOLECULAR
CLONING
NEUROFIBROMATOSIS DOMAIN
RELATED
TYPE
OF A cDNA CODING
1 PROTEIN
ISOFORM
TO ras GTPase-ACTIVATING
984-990
FOR LACKING
THE
PROTEIN’
Hiroyuki Suzuki, Kazuhiro Takahashi,Yasuhiko Kubota and Shigeki Shibahara* Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980, Japan Received
August
1, 1992
Summary Neurofibromatosis type 1 (NFl) is an autosomal dominant neurocutancous disorder, and a genelinked to NFl was recently identified. Its gene product (NFl protein) contains a domain functionally related to mammalianras GTPase-activating protein (GAP). Here, we cloned a cDNA coding for NFl protein isoform lacking the region related to GAP from a oligo(dT)-primed cDNA library of humanplacenta. This cDNA carries the insert of about 2.4 kb, coding for a protein of 551 amino acid residues,which sharesthe sameamin@ terminal 547 residueswith authentic NFl protein. We show that NFl mRNAs of about 2.9, 11, and 13 kb are expressedin human tissues,and that the isolated cDNA may represent the 0 1992ncademlc Press,1°C. 2.9-kb transcript.
Neurofibromatosistype 1 (NFl) or von Recklinghausen’sdiseaseis an autosomaldominant disorder,affecting oneper 3000-4000individuals, and is characterized by the presenceof cafk au lad skin spots,multiple neurofibromas, and Lisch nodule (1,2). Recently, a gene linked to NFl was identified by positional cloning (3-Q and structural analysis of its gene transcript revealed an open reading frame of 2818 amino acids (6). A portion of NFl protein sequence showssimilarity to mammalianras GTPase-activating protein (GAP) and yeast IRA proteins (7,8), which are known to down-regulatemammalianrasand yeast RAS proteins, respectively (9,lO). The GAP-related domainof NFl protein wasthen shownto possessGAP activity (ll13). Furthermore,NFl protein was shownto be involved in the negative control of rasin intact cells and in the inhibition of cell proliferation (14), suggestingthat the NFl gene is a tumorsuppressorgene (14-16). NFl gene product was also termed neurofibromin (17), since it is abundantin the nervous system. Recently, we and other group reported the presenceof two types of NFl mRNAs probably produced by alternative splicing; type I is authentic NFl mRNA and type II has the 63-bp isequence data from this article have beendepositedwith the EMBWGenBank Data Libraries underAccessionNo. D 12625. *To whom correspondenceshouldbe addressed. Abbreviations: NFl, Neurofibromatosis type 1; GAP, ras GTPase-activating protein; kb, kilobases;PCR, polymerasechain reaction; bp, basepairs. 0006-29 1X/92 $4.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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insertion in the GAP-related domain (1819).
Interestingly, expression of these two types of mRNAs may change during cell growth status (18), although the exact function of authentic NFl protein remains to be elucidated.
To establish the function of NFl gene product, we
attempted to clone a full-length NFl cDNA from human cDNA libraries. We thus isolated a cDNA for NFl protein isoform lacking the GAP-related domain. Its deduced protein, consisting of 551 amino acid residues, shares the same amino-terminal authentic NFl protein, except for four residues at its carboxy-terminus.
547 residues with
We then confirmed the
expression of the NFl gene transcripts coding for this NFl protein isoform in human tissues. Furthermore, we cloned a partial cDNA coding for the 3’-portion of authentic NFl mRNA, providing the first evidence that authentic NFl mRNA contains the 3’-untranslated about 3 kb.
Materials
region of
and Methods
Preparation ofRNA - Total RNA was prepared from human kidney, placenta and HeIa cells by guanidium thiocyanate-CsCl method (20). Poly(A)+ RNA was isolated by oligo (dT)cellulose chromatography (21). cDNA cloning for NFl protein - A cDNA library was constructed from 4 pg of poly(A)+ RNA of human placenta by the method of Okayama and Berg (22,23). A cDNA library of a human basophilic leukemia cell line (KU-812-F) was similarly constructed as described (24). Total plasmid DNA (1Opg) of each cDNA library was linearized with SalI, electrophoresed on agarose gels, transferred to a nylon membrane filter (Zeta-probe membrane, Bio-Rad), and hybridized with each of sap-labeled cDNA fragments generated by polymerase chain reaction (PCR). The DNA probes used were amplified from kidney cDNAs using synthetic PCR primers designed to cover the regions coding for the amino-terminus and carboxy-terminus of NFl protein (7). Synthesized were two sense primers, AA’ITGGGAAGATAACTCTGT (nucleotide numbers 1003/1022) and GTAGCCACAGGTCCGGCTCTC (6271/6290), and two anti-sense primers TGAGGGCTTATACGAAAGCA (complementary to 1147/1166) and TITAAGCAACTCTCAAGTGC (complementary to 6760/6779). The nucleotide residues were renumbered from the A residue of the initiating ATG codon (6). The amplified cDNA fragments were labeled with [a-32P]dCTP (3000 Ci/mmol, Amersham) by the method of Feinberg and Vogelstein (25). According to the size of hybridizable bands detected, the target cDNA fragments were purified, self-ligated, and used to transform E. coli DHScr. Screening such size-fractionated cDNA libraries enriched with the target clones, two different clones were isolated, and designated pHN-3 and pHN-2, representing the cDNA clones derived from placenta and a leukemia cell line, respectively. Nucleotide sequence was determined by the dideoxy chain-termination method (26) using Sequenase (USB) and BcaBEST sequencing kit (Takara). Northern blot and Sl nuclease-mapping analyses - Poly(A)+ RNA, denatured with glyoxal/dimethyl sulfoxide, was electrophoresed on an 1.0% agarose gel in 10 mM sodiumphosphate buffer (pH 7.0) (27) transferred to a nylon membrane filter (Zeta-probe membrane, Bio-Rad), and fixed with UV-linker (Stratalinker 1800, Stratagene). The HincII/PstI fragment (196/l 113) of pHN-3 was inserted into pBluescript KS vector (Stratagene), and a 32P-labeled riboprobe was prepared according to the method of Melton et al. (28). Hybridization was carried out in the mixture of 1.5x SSPE (0.225M NaCl, 15mM NaH2P04, 1.5mM EDTA, pH7.4) 50% formamide, 1% SDS, 0.5% skim milk, 0.5 mg/ml salmon testis DNA, and 0.2 mg/ml yeast tRNA. The hybridized filters were extensively washed at 50°C with 2x SSC, 0.1% SDS, and with 0.1x SSC, 0.1% SDS. The washed filters were then rinsed in 2x SSC, treated with RNase A (l&ml) in 2x SSC for 7 min at room temperature, washed in 0.1x SSC, 0.1% SDS at 50°C for 30 min, and then subjected to the detection with Bioimage Analyzer (Fuji BAS2000). Sl nuclease-mapping analysis was carried out as described previously (29). The Sl probe was prepared from a subclone carrying the ApaI/BamHI fragment of pHN-3 (1548/vector) in the SmaI site of pUC119. Both ends were converted to blunt-ends prior to 985
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ligation, and the BamHI site is located in the Okayama-Berg vector (23). Total RNA (20 pug) was hybridized with the HhaI/NcoI fragment (vector/1796) derived from this subclone,which was end-labeledat the NcoI site with [Y-3zPJATP (7000 Ci/mmol, ICN). Hybridized nucleic acids were digested with 20 units of Sl nuclease(Boehringer-Mannheim) and the protected fragmentswere analyzedon a 5% polyacrylamidegel containing 7 M urea. Radioactive signals were detectedby exposingthe dried gel to X-ray films (X-AR5, Kodak) at -85°C.
Results and Discussion Isolation aud characterization of NFl cDNA clones- To confirm the presenceof NFl cDNA clonesprior to screening,we analyzed total plasmidDNA derived from several human cDNA libraries. We thus detecteda band of about 5.5 kb with the 5’-probe in total plasmid DNA linearizedwith SalI, derived from a placentacDNA library, and detecteda 9-kb signal with 3’probe in leukemia cell cDNAs (data not shown). Since thesepositive fragments include the vector sequenceof about 3 kb (23) the actualsizesof thesecDNAs are smaller than that of the published NFl cDNA of more than 9 kb (6). We thus isolated two different cDNA clones, designated pHN-3 and pHN-2, from the size-fractionated cDNA libraries of placenta and leukemiacells, respectively (Fig.1). It shouldbe noted that thesecloneswere isolatedfrom the oligo(dT)-primed cDNA libraries and thus contain the 3’-untranslated region including a poly(A)-tail. H lkb
A NFl
mRNA
3
NF1 cDNA
I
pHN-3
,
PHN-2
I
Northern
probe
(v
I
zix c---B* Sl
probe
Figure 1. Schematic representation of NFl mRNA and its isolated cDNAs. (A) Relationship between NFl mRNA and isolated cDNAs. NFl mRNA is schematically drawn; box represents the protein-coding regionpublished (6), and solid lines, 5’ and 3’untranslated regions determined in this study. A slashed box indicates the GAP-related domain shown as GRD, and stippled boxes indicate the regions related to IRA. Cloned cDNAs for NFI mRNA are also drawn; closed boxes represent the protein-coding regions and solid lines, 5’ and 3’-untranslated regions. Two cDNAs cloned in this study are shown below the published sequence indicated as NFl cDNA. Note that the nucleotide sequence near the 3’-end of pHN-3 differs from the published sequence (6) (see Fig.2). The 5’-end of the insert cDNA carried by pHN-2 is located in the region near the nucleotide residue 5700 (indicated with two vertical lines). (B) Restriction map of pHN-3, showing only relevant or unique sites. The probes used in Fig.3 are also indicated. The Sl probe was the HhaI/NcoI fragment (vector/1796), endlabeled at the Ncol site (indicated by an asterisk); a dotted line represents the sequence of pUC119 and a solid line represents the protected fragment.
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ATCTCTAGCTCSCTCGCGCTCC TCTCCCCGGGCCGTGGAAAGGATCCCACTTCCGGTGGGGTGT~ATGG~GG~GT~T~GGA~TGTGATGG~TGTGGGGAGA~GG~G~TAGT
-361
CTTGCCAACGCCCCCTTTCCC
-181
-271
TCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACA~TGGGAGC~TG~A~T~~A~AGA~~~T~T~~TTG~~T~TT
-91
CCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGC~~ACCCTT~~~T~~G~CG~~~~~~GG~~G~GGGGAGGA~
-1
ATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAG~T~~~AATAAAAA~AGGA~AG~AGAA~A~A
90 30
MAAHRPVEWVOAVVSRFDEOLPI KTGOONT CATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACT
180 60 270
HTKVSTE HNKECLINI SKYKFSLVISGLTT ATTTTAAAGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTG I L K N VNNMRI FGEAAE K N L Y LSOLIIL GAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTT E K C LAGOPKD TMRLDETMLVKOL L P E CTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTC L
H
T
C
R
E
G
N
0
H
A
A
E
L
R
N
S
A
S
G
V
L
F
S
L
S
D
T
L
I
C
H
F
90 360 120
C
N
N
F
450 150
E
L
L
AATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTA STRLDELTVCSEDNVDVHDI NAVFSRI
540
CAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTA 0 Y I NVDCAKLKRLL K E T A F KFKALKKVAOL GCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACT~TACCAGATCCCACAGACT
210 720 240 610
AVINSLE KAFWNWVENYPDEFT KLYOIPOT GATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAA VDGFAESTKR KAAVWPLO D M A E C A E K L F D L
210 900 300
ATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTG IILLILCPEIIDDI SKDVVDENNMNKKLFL GACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCA RKALAGHGGS A C V K D S L R 0 L TESAAI
Q90 L
C
K
A
L
F
N
P
360
N
1 I10 390
AGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGC?TTTTAATCCA S
T
Y
I
N
W
E
D
N
S
V
I
FLLVOSMVVD
L
K
N
180 830
L
AGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAAC S K P F SRGSOPADVDLMI DCLVSCFRI
S
P
H
330 1060
AACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAAT NOHFKI CLAONSPSTFHYVLVNSLHRIITN TCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCA
1260 420 1360
S A L DWWPKIDA VYCHSVE LRNMFGETLHKA GTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGC~CCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAA R M A-P S L T F KEKVTSLKFKEK VOGCGAHPAI
450 I440 400
CCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCAT~GTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTTGTAAT SYKYLLLSM-VKLI HADPKLLLCN PTDLETR
1530 510
CCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAG TOGSTAELITG LVOLVPOSHMPE P RKOGPE
1620 540 1710
ATTGCTCAGGAAGCAATGGAGG~AAGGGGAAAATGAATTCCATGTTCTTGAAGGAAAGACTGTAACTATGTACATTCATGATGTTCCTTTI IAOEAME R G K t
551
GTGTGTGGTTTCTGTGAGTAACAGGTAGATGTCATTTCTGGAAATGGTATGTTTATGTCTATACATTGTTTTATAAAACTCCATGGAGA AGAAGGGGTTTACTTGCTTTGTATCACATAGCAATAACATTGTACAAATTCTGATGCTTAATAAAATAGTTCGAGATTTTC
1000 (A)n
1882
Figure 2. Nucleotide sequence of a cDNA coding for NFl protein isoform and its deduced amino acid seauence. The nucleotidc seauencc of DHN-3 is shown and is numbered in the 5’ to 3’ direct& from the A residue of the&initiating methionine codon ATG. The nucleotides on the 5’ side of residue 1 are indicated by negative numbers. The deduced amino acids are shown below the nucleotidc sequence and arc numbcrcd beginning with the initiating methionine. Boxed are the sequences newly identified. Double underlines indicate base changes at 702, 1389, and 1488. A putative poly-adcnylation signal is underlined, and a poly(A)-tail is shown as (A)n.
pHN-3 carriesthe cDNA insert of only about 2.4 kb, although pHN-3 was isolated by using the S-probe. The nucleotidesequenceof its cDNA insertwas then determined(Fig.2). pHN-3 contains the same5’-untranslatedregion asthe publishedNFl cDNA except that its length is longer by about 168 bp than that of the publishedNFl cDNA. The initiating methionine codon nucleotideresiduesl-3) was assignedaccording to the one of the authentic NFl cDNA reportedby Marchuk et al. (6). The nucleotidesequencefrom the initiation codon to the residue (ATG,
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1642 is identical to the sequence published previously, except for three bases as indicated in Fig.2. The nucleotide sequence downstream from position 1643 is different from that of NFl cDNA, and a codon for termination (TGA) is present at 1654/1656. Thus, pHN-3 contains the open reading frame of 1653 nucleotides, coding for 551 amino acid residues with a molecular weight of 62,300. The protein encoded by pHN-3 contains the four amino acid residues at its carboxyl terminus, which are not present in authentic NFl protein.
It is remarkable that this
isoform lacks the segment coding for GAP-related or IRA-related region (Fig.1). Since the nucleotide sequence AG/GTAAG (1640/1646) conforms to the consensus sequence of the donor site for splicing (30), it is conceivable that a part of the sequence downstream from 1642 may be spliced out as an intron for some NFl gene transcript. Three base changes were found in pHN-3 as described above and may represent polymorphism. residue 1488 leads to the amino acid substitution;
One base change at nucleotide
namely, pHN-3 contains the codon ATG
(1486-1488) for Met at 496, while the published cDNA contains ATA codon for Ile (7). This base change was also found in other NFl cDNA (6). Two base changes, leading to no amino acid substitutions, are found at nucleotide residues 702 and 1389; pHN-3 contains the A residue at both sites, while the published cDNA contains G and C residues, respectively (6,7). pHN-2 was characterized by restriction endonuclease mapping and a part of its insert DNA was sequenced. pHN-2 carries a cDNA of about 6 kb, coding for the carboxy-terminal portion of authentic NFl protein and the 3’-untranslated
region of about 3 kb. This size of the 3’-
untranslated region is similar to its assumed length of about 4 kb (6).
We thus propose that
authentic NFl mRNA contains the 3’-untranslated region of about 3 kb. Expression of the mRA4 codittg for NFl protein isoform - We investigated the expression of the transcripts coding for NFl protein isoform by Northern blot analysis. The cRNA probe used contains the region which is common to the transcripts coding for authentic NFl protein and its isoform (see Fig.1). We were thus able to detect at least three species of mRNAs about 13, 11, and 2.9 kb in both kidney and placenta poly(A)+ RNA (Fig.3A).
of
It seems likely
that the NFI mRNA of 13 or 1I kb may contain the 3’-untranslated region of 3 kb because the size of published cDNA sequence is more than 9 kb (6). This is the first report showing multiple species of NFl mRNAs detected by Northern blot analysis, be generated by alternative splicing. To confirm the expression represented by pHN-3, we designed Sl mapping analysis (Fig.3B).
These NFl mRNAs may of the 2.9-kb NFL mRNA The protected fragment of
expected size (249 bases) was detected in kidney, placenta and HeLa cell RNA, indicating that the 2.9-kb mRNA is indeed expressed. It remains to be elucidated whether the amino-terminal NFl protein isoform is actually expressed. To answer this question, we are currently producing antibodies against the amino-terminal domain of NFI protein. Implications for thepresence ofNF1 protein isoform - NFl protein is a molecule of 280 kDa (31), but its functional domain is determined only in the GAP/IRA-related region which is located in the center of the molecule. The function of other regions of NFl protein, including the amino-terminal domain, remains to be elucidated. In present paper, we suggest the existence of a NFl protein isoform possessing only amino-terminal portion of NFl protein and lacking GAP/IRA-related domain. Thus, it seemsunlikely that such an isoform could function as an 988
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Figure 3. Expression of the 2.9-kb mRNA coding for NFl protein isoform. (A) Northernblot analysis.Eachlanecontained 2.5p”gof poly(A)+RNA preparedfromhuman
kidney or placenta. A closedarrowheadindicatesthe transcriptsof about2.9 kb. A small horizontalbar indicatesthe origin of lanes. (B) Sl nuclcasemappinganalysis. Eachlane containedthe protectedproductswith yeasttRNA or total RNA preparedfrom the indicated sources.An openarrowheadindicatesthe Sl probeof 425bases,anda closedarrowhead indicatestheprotectedfragments, representing the1.9-kbmRNA. The sizemarkersusedwere theend-labeled 0X-174DNA digestedwith HacIII, andgivenin bases. upstreamregulator for ras protein. However, if the amino-terminal domain of NFl protein is involved in binding to somecellular protein, this NFl isoform may competewith NFl protein for binding to sucha protein, leadingto the altered expressionof GAP-like activity of authentic NFl protein. Alternatively, such an interaction involving this isoform may result in operating the ras signal transduction system in a manner that is independent of ras protein. These possibilitiesare consistentat leastin part with a model that NFl protein is a downstreameffector of ras protein (32,33). Elucidation of the function and regulation of this isoform may help to understandthe role of NFl protein in cellular signaltransductionsystemand the molecularbasis of the diverse clinical manifestationsof NFl. Acknowledgments This work was supportedin part by a Grant-in-Aid for Encouragementof Young Scientiststo H.S. from the Ministry of Education, Scienceand Culture of Japan,andby a grant to S.S. from the TakedaScienceFoundation. References
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