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• W2000763279 abstract "Fibroblast growth factor (FGF) homologous factors-1, -2, -3, and -4 (FHFs 1–4; also referred to as FGFs 11–14) comprise a separate branch of the FGF family and have been implicated in the development of the nervous system and limbs. We report here the characterization of multiple isoforms of FHF-1, -2, -3, and -4 which are generated through the use of alternative start sites of transcription and splicing of one or more of a series of alternative 5′-exons. Several isoforms show different subcellular distributions when expressed in transfected tissue culture cells, and the corresponding differentially spliced transcripts show distinct expression patterns in developing and adult mouse tissues. Together with the evolutionary conservation of the FHF isoforms among human, mouse, and chicken, these data indicate that alternative promoter use and differential splicing are important regulatory processes in controlling the activities of this subfamily of FGFs. Fibroblast growth factor (FGF) homologous factors-1, -2, -3, and -4 (FHFs 1–4; also referred to as FGFs 11–14) comprise a separate branch of the FGF family and have been implicated in the development of the nervous system and limbs. We report here the characterization of multiple isoforms of FHF-1, -2, -3, and -4 which are generated through the use of alternative start sites of transcription and splicing of one or more of a series of alternative 5′-exons. Several isoforms show different subcellular distributions when expressed in transfected tissue culture cells, and the corresponding differentially spliced transcripts show distinct expression patterns in developing and adult mouse tissues. Together with the evolutionary conservation of the FHF isoforms among human, mouse, and chicken, these data indicate that alternative promoter use and differential splicing are important regulatory processes in controlling the activities of this subfamily of FGFs. fibroblast growth factor fibroblast growth factor homologous factor rapid amplification of cDNA ends polymerase chain reaction nuclear localization signal endoplasmic reticulum group of overlapping clones. Each FHF transcript or protein is indicated by a species designation consisting of a lower case letter preceding FHF (c, chicken human mouse) and a 5′-splice isoform designation consisting of “1” followed by a letter in parentheses after FHF. For example, mFHF-2 (1Y+1V) refers either to the mouse FHF-2 transcript carrying exons 1Y and 1V at its 5′-end or to the protein encoded by that transcript Members of the fibroblast growth factor (FGF)1 family of signaling proteins play diverse roles in numerous developmental processes (for reviews, see Refs. 1.Gosporadowicz D. Curr. Top. Dev. Biol. 1990; 24: 57-93Crossref PubMed Scopus (134) Google Scholar, 2.Martin G.R. Development. 1998; 12: 1571-1586Google Scholar, 3.Szebenyi G. Fallon J.F. Int. Rev. Cyt. 1999; 185: 36-45Google Scholar). Previously we described the identification of four novel FGFs, referred to as fibroblast growth factor homologous factors (FHF-1, -2, -3, and -4; now equivalently called FGF-12, -13, -11, and -14, respectively) which are prominently expressed in the nervous system in mice and chickens (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar, 5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar). The FHFs form a distinct branch of the FGF family and show 30–50% amino acid sequence identity with other FGFs. Within the FHF subfamily, different members share 60–70% identity. We and others have reported the identification of several alternatively spliced FHF transcripts in mice and chickens which code for FHF proteins with distinct amino termini (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar, 6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar, 7.Yamamoto S. Mikami T. Ohbayashi N. Ohta M. Itoh N. Biochim. Biophys. Acta. 1998; 1398: 38-41Crossref PubMed Scopus (22) Google Scholar). In chicken embryos the two previously identified FHF-2 transcripts have distinct and nonoverlapping spatial distributions, suggesting that the encoded proteins may have distinct roles during development (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar). To explore the extent to which the diversity and expression of the FHFs are regulated by differential promoter use and/or splicing of 5′-exons, we searched for novel FHF transcripts that differ at their 5′-ends. This paper reports the identification and characterization of a large variety of FHF isoforms that are conserved among human, mouse, and chicken. These isoforms (three in the FHF-1 gene, nine in the FHF-2 gene, and two each in the FHF-3 and FHF-4 genes) code for proteins with highly divergent amino termini. When expressed in cultured cells, several of the isoforms have different subcellular distributions. Analysis of the expression of the differentially spliced coding exons of each of the FHFs by RNase protection and in situhybridization in the developing and adult mouse shows that many isoforms have distinctive patterns of expression. Taken together, these observations imply that alternative promoter use and alternative splicing of 5′-exons are important for the regulation of FHF gene expression and for the generation of FHF proteins with distinct biochemical properties. Double-stranded e15 mouse embryo cDNA (CLONTECH) was used as template for 5′-rapid amplification of cDNA ends (RACE; 8). Two sequential PCRs were performed with two pairs of nested adaptor primers and gene-specific primers located in exon 2, and the resulting PCR products were resolved by agarose gel electrophoresis, cloned into pBS-KS or λgt10, and sequenced. An e10 chicken brain cDNA library (a gift of Dr. M. Tessier-Lavigne) was screened with probes derived from: (a) the alternatively spliced isoforms obtained in the mFHF-2 5′-RACE PCR; (b) exon 1S of hFHF-2; and (c) exons 2–5 of mFHF-4. A chicken e6 retina cDNA library (a gift of Dr. L. Gan) and a chicken stage 21–22 limb cDNA library (a gift of Dr. J. F. Fallon) were screened with probes derived from exons 2–5 of mFHF-1. These screens yielded cDNAs encoding the complete coding regions of cFHF-1(1B), cFHF-2(1Q+1Y′), cFHF-2(1Q+1Y′+1V), and cFHF-4(1B), and a cDNA encoding cFHF-4(1A) lacking the first 10 codons. Filters were hybridized in 30–40% formamide, 5 × SSC at 42 °C, and washed in 2 × SSC at 50 °C-55 °C. The chicken FHF-2(1U) isoform was amplified by degenerate PCR using chicken genomic DNA as template and the following primers (containingBamHI and EcoRI sites at the 5′- and 3′-ends): 5′-GCTCATGGATCCAGTA(C/T)G(A/G)GCTTGATGTTTCTGGCC-3′ (sense primer) and 5′-GCTCATGAATTCCTGAATA(C/T)GACTTCCTTAACAAAGCC-3′ (antisense primer). PCR conditions were as follows: (1×) 94 °C, 7 min; (10×) 94 °C, 30 s; 95 °C, 15 s; 45 °C, 2 min; 72 °C, 30 s; (20×) 94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min; (1×) 72 °C, 10 min. The PCR product was digested and subcloned into pBS-KS (Stratagene) and sequenced. Rabbit polyclonal anti-FHF-2 antibodies were raised against a bacterial fusion protein consisting of the entire FHF-2 open reading frame fused to the T7 gene 10 protein (9.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorf J.W. Methods Enzymol. 1980; 185: 60-89Crossref Scopus (6006) Google Scholar). Anti-FHF-2 antibodies were affinity purified using the fusion protein immobilized onto nitrocellulose; those antibodies directed against the fusion partner were removed by absorption to immobilized T7 gene 10 protein. By Western blotting and by staining of transfected cells, the antibodies purified in this fashion did not cross-react with FHF-1 or FHF-4 proteins. Anti-FHF-1 antibodies are described in Ref. 4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar. cDNA segments encoding FHF-2(1S), FHF-2(1U), FHF-2(1V), FHF-2(1Y), and FHF-2(1Y+1V) were subcloned into the pCIS vector (10.Gorman C.M. Gies D.R. McCray G. DNA Prot. Eng. Tech. 1990; 2: 3-10Google Scholar) for eukaryotic expression. To increase the efficiency of translation, the region immediately upstream of the putative initiator methionine was converted to an optimal ribosome binding site (CCACCATG) by PCR mutagenesis. The transfected cDNAs encode FHF-2 isoforms from the following species: FHF-2(1Y+1V), chicken; FHF-2(1Y), mouse; FHF-2(1V), mouse; FHF-2(1S), human; FHF-2(1U), human. Exons 2–5 of human and mouse FHF-2 encode protein sequences that differ by a single amino acid substitution; the corresponding human and chicken sequences differ by five amino acid substitutions. Expression in transiently transfected 293 cells was assessed by Western blotting with anti-hFHF-2 polyclonal antibodies. Transient transfection and immunostaining were performed as described in Ref. 4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar. The two isoforms of FHF-4, FHF-4(1A) and FHF-4(1B), were subcloned into pCIS, with an optimal ribosome binding site at the initiator codon, and tagged at the COOH terminus with a myc tag (11.Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar). The FHF-4(1A) isoform was from mouse, and the 1B isoform was from chicken. Exons 2–5 of mouse and chicken FHF-4 differ by 11 amino acid substitutions. These proteins were produced in transiently transfected COS and 293 cells, and their expression levels assayed with anti-myc monoclonal antibody 9E10 (11.Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar). 20-μm frozen sections were cut from e12.5 embryos, e18.5 embryos, and adult mouse brains and hybridized with probes derived from exons 2–5 of mFHF-1, mFHF-2, mFHF-3, and mFHF-4. The sections were also hybridized with probes derived from the following alternate mouse exons: mFHF-1, exon 1A; mFHF-2, exons 1S, 1Y, 1V, and 1U; mFHF-3, exons 1A and 1B; mFHF-4, exons 1A and 1B. 33P-Labeled probes were prepared byin vitro transcription of PCR-amplified templates containing the T3 promoter in the antisense primer. In situhybridization was performed essentially as described in Ref. 4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar, with the addition of 10 mm Na-EDTA to all buffers. Autoradiographic images and cresyl violet-stained tissue sections were scanned, and the images were merged and manipulated using Photoshop 5.0 software for the Macintosh. RNase protection experiments were carried out using an Ambion RPA II kit following the manufacturer's instructions. In brief, individual mFHF exons were subcloned into pBS-KS and transcribed in vitro using T3 or T7 polymerases with an Ambion Maxiscript kit. 4 × 105 cpm of32P-labeled probes was used per reaction with 10 μg of total RNA isolated from the following adult mouse tissues: brain, eye, heart, liver, lung, kidney, spleen, and testis. 10 μg of yeast tRNA was used as a control. We and others (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar, 6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar, 7.Yamamoto S. Mikami T. Ohbayashi N. Ohta M. Itoh N. Biochim. Biophys. Acta. 1998; 1398: 38-41Crossref PubMed Scopus (22) Google Scholar) have reported the identification of several isoforms of FHF-1, FHF-2, and FHF-4 in which distinct amino termini are generated by differential splicing. Our initial characterization of transcripts encoding the cFHF-2(1Y+1V) and cFHF-2(1S) isoforms in chicken embryos showed that they are expressed differentially in the developing neural tube (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar). Differential expression of two mFHF-4 splice isoforms was reported in the adult rat brain (7.Yamamoto S. Mikami T. Ohbayashi N. Ohta M. Itoh N. Biochim. Biophys. Acta. 1998; 1398: 38-41Crossref PubMed Scopus (22) Google Scholar). These observations, together with the high degree of conservation of the two alternate FHF-2 exons between humans and chickens, suggested that alternative splicing of the FHFs may be a general and conserved mechanism for regulating FHF gene expression. To investigate whether there are other splice isoforms of the FHFs with divergent amino termini, we screened cDNA libraries from mouse and chicken embryos, brain, and retina and performed 5′-RACE with e15 mouse embryo RNA. We also examined human brain and retina cDNA clones from earlier screens for evidence of alternative splicing. This search identified three isoforms of FHF-1, nine isoforms of FHF-2, and two isoforms each of FHF-3 and FHF-4 (TableI). Results from 5′-RACE suggest that in the mouse embryo, FHF-2 and FHF-3 display the largest number of distinct transcripts. In contrast, FHF-1 and FHF-4 5′-RACE reactions with mouse embryo RNA revealed only a single product in each case, and these were identified as the mFHF-1(1A) and mFHF-4(1B) isoforms. Thus far, one novel mFHF-3 isoform has been identified from the 5′-RACE reaction, mFHF-3(1B).Table IDifferentially spliced FHF exons identified in human, mouse, or chicken cDNAGene5′-exonsSpeciesSourceFHF-11AHumanSmallwoodet al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar).Mousee15 mouse embryo RACE; Smallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar); Hartung et al. (6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar).Chickene10 brain, e6 retina chick cDNA libraries; Munoz-Sanjuan et al. (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar).FHF-11BHumanHuman brain cDNA library.MouseHartung et al. (6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar).Chickene10 chick brain cDNA library.FHF-11CMouseHartung et al. (6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar); incomplete cDNA.FHF-11DChickenStage 21–22 limb library; no initiator Met found.Reverse transcriptase PCR from stage 23–24 chick embryo.FHF-21SHumanSmallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar); Hartung et al. (6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar).MouseSmallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar); Hartung et al. (6.Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar).FHF-21T + 1S′Chickene10 chick brain cDNA library.FHF-21P + 1Y + 1VMousee15 mouse embryo RACE.FHF-21Q + 1Y′ + 1VChickene6 retina and e10 brain chick cDNA libraries; Munoz-Sanjuan et al. (5.Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar).FHF-21Q + 1Y′Chickene6 retina and e10 brain chick cDNA libraries.FHF-21RMousee15 mouse embryo RACE; incomplete cDNA.FHF-21UHumanHuman fetal heart, ESTsAA449030 and AA427960; Gecz et al. (16.Gecz J. Baker E. Donnelly A. Ming J.E. McDonald-McGinn D.M. Spinner N.B. Zackai E.H. Sutherland G.R. Mulley J.C. Human Genetics. 1999; 104: 56-63Crossref PubMed Scopus (70) Google Scholar).Mousee15 mouse embryo RACE.ChickenDegenerate genomic PCR.FHF-21X + 1W + 1VHumanHuman brain cDNA library.FHF-21Z + 1YHumanHuman brain cDNA library.FHF-31AHumanSmallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar).MouseSmallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar).FHF-31BMousee15 mouse embryo RACE.FHF-41AHumanSmallwood et al. (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar).Mousee15 mouse embryo RACE; Smallwood et al.(4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar).Chickene10 chick brain cDNA library.FHF-41BMousee15 mouse embryo RACE; Yamamotoet al. (17.Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (400) Google Scholar).Chickene10 chick brain cDNA library.Each class of FHF transcript is listed on a separate line, together with the species from which the corresponding cDNA and/or RACE PCR product was derived. Some transcript classes include more than one exon upstream of exon 2; these are listed in the table in the order in which they are spliced together (5′ to 3′). In each class, the 3′-most exon listed is spliced to the second exon of the indicated FHF. References are listed for those exons that have been reported previously. Open table in a new tab Each class of FHF transcript is listed on a separate line, together with the species from which the corresponding cDNA and/or RACE PCR product was derived. Some transcript classes include more than one exon upstream of exon 2; these are listed in the table in the order in which they are spliced together (5′ to 3′). In each class, the 3′-most exon listed is spliced to the second exon of the indicated FHF. References are listed for those exons that have been reported previously. One cFHF-1 cDNA clone isolated from a chicken limb cDNA library (isoform 1D; Table I) does not appear to encode a functional open reading frame unless translation is initiated from a non-AUG codon, as is the case for some FGFs (12.Bugler B. Amalric F. Prats H. Mol. Cell. Biol. 1991; 11: 573-577Crossref PubMed Scopus (304) Google Scholar, 13.Acland P. Dixon M. Peters G. Dickson C. Nature. 1990; 343: 662-665Crossref PubMed Scopus (214) Google Scholar, 14.Florkiewicz R.Z. Sommer A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3978-3981Crossref PubMed Scopus (447) Google Scholar). The existence of this isoform was verified independently by reverse transcriptase PCR from stage 23–24 chicken embryo RNA. FHF-2 shows the greatest diversity of spliced isoforms, with nine different transcripts identified in various species (Table I). The majority of the FHF-2 transcripts were identified either from mouse or chicken cDNAs, but most of the alternate exons also have counterparts in human genomic DNA. All of the homologous human sequences are present at the FHF-2 locus 5′ of the invariant exons (exons 2–5) and in the same orientation as those exons. For example, exon 1U was identified initially as part of a mouse 5′-RACE product and was found subsequently in the chicken genome by PCR, in human genomic DNA sequences from the FHF-2 locus, and in an expressed sequence tag from developing human fetal heart. One cDNA segment from the mouse 5′-RACE analysis, exon 1R, is highly homologous to a small segment of human genomic DNA between exons 1S and 2 of the human FHF-2 gene. Although this exon has not been found in any human cDNA, its location within the human FHF-2 gene suggests that it forms a part of that gene. All of the alternate 5′-exons that contain coding sequences (1S, 1U, 1V, and 1Y) have been identified in human, mouse, and chicken and are highly conserved (Table II).Table IIFHF isoforms generated by 5′ alternative splicingExonsSequenceSpeciesSubcellular localizationFHF-11AMAAAIASSLIROKROARESNSDRVSASKRRSSPSKDGRSLCERHVLGVFSKVRFCSGRKRPVRRRP/EPQL …HumanNuclear––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––/EPQL …MouseAbsent in nucleoli––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––/EPQL …Chicken 1BMESK/EPQL …HumanCytosolic + nuclear––––/EPQL …Mouse––G–/EPQL …ChickenFHF-21SMAAAIASSLIRQKRQAREREKSNACKCVSSPSKGKTSCDKNKLNVFSRVKLFGSKKRRRRRP/EPQL …HumanNuclear + nucleoli––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––/EPQL …Mouse––––––––––––––––––––––––––––N––––S–G––––––––––––––––––––––––––/EPQL …Chicken 1UMALLRKSYS/EPQL …HumanCytosolic + nuclear–––––––––/EPQL …Mouse–––––––––/EPQL …Chicken 1VMSGKVTKPKEEKDASK/EPQL …HumanCytosolic + nuclear––––––––––––––––/EPQL …Mouse–––––I––––––––––/EPQL …Chicken 1YMLRQDSIQSAELKKKESPFRAKCHEIFCCPLKQVHHKENTEPE/EPQL …HumanCytosolic + nuclear––––––––––––––––––––––––––––––P––––––––––––/EPQL …MouseMDDAPPGTQEYI–––––––––––––––––––––––––––––––––––L–––––––/EPQL …Chicken 1Y + 1VMSGKVTKPKEEKDASK/VLDDAPPGTQEYIMLRQDSIQSAELKKKESPFRAKCHEIFCCOLKQVHHKENTEPE/EPQL …Human––––––––––––––––/–––––––––––––––––––––––––––––––––––––––––––P––––––––––––/EPQL …MouseCytosolic + nuclear–––––I––––––––––/–M––––––––––––––––––––––––––––––––––––––––––––––L–––––––/EPQL …ChickenFHF-31AMAALASSLIRQKREVREPGGSRPVSAQRRVCPRGTKSLCQKQLLILLSKVRLCGGRPARPDRGP/EPQL …HumanND–––––––––––––––––––––––––––––––––––––––––––––––––––––––––T–Q––––/EPQL …Mouse 1BMSLS/EPQL …MouseNDFHF-41AMAAAIASGLIRQKRQAREQHWDRPSASRRRSSPSKNRGLCNGNLVDIFSKVRIFGLKKRRLRRQ/DPQL …HumanNuclear–––––––––––––––––––––––––––––––––––––––F–––––––––––––––––––––––/DPQL …MouseAbsent in nucleoli–––––––––––––––––––N––––––––C–––––––––––––––––––––––/DPQL …Chicken 1BMVKPVPLFRRTDFKLLLCNHKGLFFLRVSKLLGCFSPKSMWFLWNIFSKGTHMLQCLCGKSLKKNKNPT/DPQL …MouseCytosolic–I–––––––––GLN–––––R–D––––––Y–––D–––––––––––––––––S––––––––––––––––Q–/DPQL …ChickenAmino acid sequences of the alternative amino termini for the various FHF isoforms derived from human, mouse, and chicken. A slash indicates the junction between exons. The first four amino acids encoded by the invariant exon 2 are indicated at the end of each sequence. Subcellular localization was determined by immunostaining of protein produced in transfected cells as described in the text. ND, not determined. Underlined residues in the FHF-1(1A) isoform indicate the bipartite (left) and secondary (right) nuclear localization sequences as described in Ref. 4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar. Open table in a new tab Amino acid sequences of the alternative amino termini for the various FHF isoforms derived from human, mouse, and chicken. A slash indicates the junction between exons. The first four amino acids encoded by the invariant exon 2 are indicated at the end of each sequence. Subcellular localization was determined by immunostaining of protein produced in transfected cells as described in the text. ND, not determined. Underlined residues in the FHF-1(1A) isoform indicate the bipartite (left) and secondary (right) nuclear localization sequences as described in Ref. 4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar. Fig. 1 shows the complex structures of the differentially spliced 5′-ends of the FHF-2 transcripts. In several cases, the 5′-coding exons are spliced together with different noncoding exons. Within exons 1Y and 1S, some splicing events retain only the 3′-regions of these exons (termed 1Y′ and 1S′, respectively) as parts of longer transcripts. Several of the FHF-2 5′-exons reported here are noncoding because there are stop codons in all three reading frames (exons 1P, 1Q, 1T, 1W, 1X, and 1Z). Most of the putative 5′-coding exons contain only one potential initiator methionine codon, and, except for exon 1U, the sequence around that methionine is not a close match to the optimal consensus for the initiation of translation (15.Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). In one instance (exon 1V) there is a potential initiator methionine codon in chicken cDNA which is absent from the human and mouse cDNAs (AUG in chicken versus CUG in mouse and human; see Table II). In this regard, it may be relevant that some FGF-2 and FGF-3 translation products are initiated at CUG codons (12.Bugler B. Amalric F. Prats H. Mol. Cell. Biol. 1991; 11: 573-577Crossref PubMed Scopus (304) Google Scholar, 13.Acland P. Dixon M. Peters G. Dickson C. Nature. 1990; 343: 662-665Crossref PubMed Scopus (214) Google Scholar, 14.Florkiewicz R.Z. Sommer A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3978-3981Crossref PubMed Scopus (447) Google Scholar). Identification of homologous spliced isoforms in the chicken and mouse FHF-2 genes suggests that the alternative 5′-exons have been conserved through evolution. The human FHF-2 gene maps to Xq26 (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar), and this region has been sequenced extensively, allowing the unambiguous identification of human counterparts for most of the exons identified in other species. Fig.2 shows the current map of the hFHF-2 gene which encompasses at least 400 kilobases of genomic DNA. Many of the alternate 5′-exons cluster within the gene, and several predicted introns are more than 100 kilobases in length. Noncoding 5′-exons 1T and 1Q have not yet been identified in humans. The nucleotide sequences of both coding and noncoding exons are highly conserved across species. For example, 5′-untranslated regions within the noncoding portions of the alternative coding exons share >90% nucleotide sequence identity between human and chicken. In earlier work, we identified a nuclear localization signal (NLS) in FHF-1 (now FHF-1(1A)), which is sufficient to confer nuclear localization in a variety of tissue culture cell lines (4.Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H.C. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (335) Google Scholar). The NLS sequence is conserved among those isoforms of FHF-2, -3, and -4 which most closely resemble FHF-1(1A), and is absent from several isoforms of FHF-1, -2, -3, and -4 which differ in their amino-terminal sequences (Table II). To analyze the subcellular distribution of the different FHFs and their various isoforms, chicken, mouse, and human FHFs were produced in transiently transfected 293, COS, and QT6 cells. Similar results were obtained with the different cell lines. As expected, FHF-2(1S), the isoform most similar to FHF-1(1A), localizes to the nucleus in transfected cells (Fig. 3 A). The FHF-2 isoforms beginning with exons 1U, 1V, 1Y, or (1Y+1V) lack a consensus NLS, and the encoded proteins show a diffuse cytosolic and nuclear distribution when transiently transfected into 293 or QT6 cells (Fig. 3 B; Table II). Double immunofluorescence staining of these FHF-2 isoforms and BiP, an ER-resident protein, reveals that they are not localized to the ER (Fig. 3 B). Their localization in the nucleoplasm may reflect passive diffusion of the FHFs through nuclear pores. In contrast, a myc-tagged version of cFHF-4(1B) is localized exclusively in the cytosol in COS and 293 cells (Fig. 3,D and E), whereas mFHF-4(1A), which has a consensus NLS, shows nuclear localization, although it is excluded from the nucleoli (Fig. 3 C). Hydropathy analysis of the FHF-4(1B) splice variant shows a hydrophobic stretch in the first 25 residues of this isoform which could act as an ER-targeting signal sequence, the only such sequence identified thus far among the FHFs. However, we have been unable to detect significant quantities of myc-tagged FHF-4(1B) or of any other FHF in conditioned media from transfected cells. The FHF-2(1S), FHF-2(1V), and FHF-2(1Y+1V) isoforms were tagged at their carboxyl termini with six histidines and purified from 293 cell lysates by affinity chromatography using nickel resin and heparin agarose. The FHF-2(1S) isoform binds heparin more tightly than the other two isoforms, eluting at 0.7 m NaCl rather than at 0.4 m NaCl as seen for FHF-2(1V) and FHF-2(1Y+1V). This observation suggests that different FHF isoforms may differ in their interac" @default.
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• W2000763279 title "Isoform Diversity among Fibroblast Growth Factor Homologous Factors Is Generated by Alternative Promoter Usage and Differential Splicing" @default.
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