FXR1 FM R FM R P FXR 1. In teraction s between Six FM RP Isoform s and FXR1 Prote in

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1 14 4 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy A ug 1998 FM RP 3 6 FXR1 A Sittler 3 3 J2L M andel 3 3 (, ; 3 3 IGBM C, CN RS2IN SERM 2UL P, Illk irch, F rance) X, FM R 1 FM R 1 FM R (FM R P) X 1 (FXR 1) FM R FM R P FXR 1, FM R 1 FXR FM R P FXR 1, C FM R P FXR 1 : X, X 1,, In teraction s between Six FM RP Isoform s and FXR1 Prote in Chen Yu2T ing A Sittler 3 3 Yu M in W u Guan2Yun J2L M andel 3 3 Shen Yan (N ational L aboratory of M ed ical M olecular B iology, Institute of B asic M ed ical S ciences, Ch inese A cad emy of M ed ical S ciences & P ek ing U nion M ed ical Colleg e, B eij ing ; 3 3 IGBM C, CN R S 2IN S ERM 2UL P, I llk irch, F rance) Abstract F ragile X syndrom e is the m o st comm on fo rm of hereditary m en tal retardation, re2 su lting from the lack of exp ression of fragile x m en tal retardation gene 1 (FM R 1) Com p lex alternative sp licing ex ists du ring FM R 1 exp ression and leads to variou s fragile X m en tal re2 tardation p ro tein (FM R P) isofo rm s,w ho se b io logical ro les are no t know n yeṫ To find ou t the effect of alternative sp licing on the fo rm ation of heterogeneou s dim er betw een FM R P and FXR 1, the in teraction s betw een six FM R P isofo rm s resu lting from alternative sp licing of ex2 on s 12, 14, 15, and FXR 1 p ro tein w as studied by the u se of the yeast tw o2hyb rid system R e2 su lts show that the strength of in teraction decreases as the C2term inu s length of the isofo rm s increaseṡ It suggests that though alternative sp licing of exon s 12, 14, 15 has no effect on the in teractiveness betw een FM R P and FXR 1, the differen t C2term inu s cou ld effect on the stab il2 ity of heterogeneou s dim er betw een FM R P and FXR 1 Key words: Yeast tw o2hyb rid system, FM R 1, Isofo rm s, FXR 1 3 (B96217), ( ) 396 X, : , :

2 X 1 (fragile X m en tal retardation gene 1, FM R 1) FM R 1 17, [ 1, m RNA ] FM R 1 (FM R P) RNA,, [ 3 RNA ] FM R P [ 4, ] FM R 1 2 FXR 1 FXR [ 5, 6, ] FM R P FXR 1 FXR 2 FM R P,, FM R FM R P, FXR 1, FM R 1 FM R P FXR 1 FM R 1 1 [7 ] 111 L 40, trp 1 leu2 h is3, h is3 lacz DNA pbtm 116 (F ig 1), lexa DNA, TR P1 pa SV 3m (F ig 2) (NL S) V P16 L EU FM R1 FXR1 K lenow T aq P rom ega Boeh ringer [ 8 M annheim FM R 1 6 iso ] F ig 1 EcoR g P stg FM R 1, EcoR g P stg pbtm 116, lexa Iso1 1 iso7 1 pn I : FM R 1 iso FM R 1 cdna, L S (aa430, 432, 435) [ 9 ] ; FM R 1 iso7 1 L S (aa430, 432, 435) FM R 1 iso7; pn I FM R P 1 10 FM R 1 iso1 1 FM R 1 cdna (EcoR g gp stg ) pbtm 116 iso7 1 Kpng P stg iso1 1 (Kpng 13 ), 14 L S (aa430, 432, 435) EcoR g Kpng FM R 1 iso7, 13 EcoR g P stg pbtm 116, 14 L S (aa430, 432, 435) FM R iso7, FM R 1 iso7 1 pn I : K sp 632 I iso1, K lenow EcoR g, FM R g 1 10, Sm ag EcoR g pbtm 116 pn İ FXR 1gpA SV 3m (F ig 2) : FXR 1 V P16, PCR FXR 1 cdna, A T G Bam H g, PCR Bam H g Sph g Sphg Bg1 g FXR 1 cdna, Bam H g Sphg PCR Bam H g Bg1 g pa SV 3m 397

3 F ig 1 Structures of the 6 different FM RP isofo rm s, pn I(upper) and DNA binding hybrid vecto r pbtm 116 (low er) U pper:w eak lines indicate new C2term inal sequences generated due to alternative sp licings a, b, c deno te the first, second and th ird sp licing dono r sites of exon 15 N um bers in brackets donate theo retical mo lecular w eigh ts in kd Low er: lexa : sequence of lexa DNA binding dom ain, TRP1: yeast selecting gene, amp: antiamp gene, o r: rep licon in E coli, P: transcrip tion p romo ter F ig 2 Structures of FXR 1 (upper) and the tran2 scrip tion activating hybrid vecto r pa SV 3m (low er) V P16: sequence of V P16 transcrip tion activating dom ain; L EU 2: yeast selecting genes; amp: anti2 amp gene; o r: rep licon in E coli; P: transcrip tion p romo ter; NL S: nuclear localisation sequence PCR [ 10 ] PEG3350 Sigm a,l ia c DM SO

4 DNA L 40, L m l L 40 (A 660nm = 018) 50 m l T E, 2 m l 100 mm o lgl L ia cg0 5 T E 100 Λl, 1 Λg 100 Λg DNA, 700 Λl 100 mm o lgl L ia cg40% PEG3350g1 T E 30 m in, 88 Λl DM SO, 42 7 m in, gλg DNA L 40 YEPD (1%, 2%, 2% ) Yc 1 L Yc 112 g Yeast n itrogen base, 5 g, 10 g, 011 g, L L 0105 g (Yeast n itrogen base Sigm a, )ph 710 FM R 1 Yc (Ycg2trp ) ; FXR 1 Yc (Ycg2leu) ; Ycg2trp, 2leu, 2u ra 2, Ycg2trp, 2leu, 2h is, 2u ra, 2lys H is d 114 Β- [ 10 ] : 5 m l Ycg2trp, 2leu, 2u ra, 30 ( ) ; , Z ( 0127% Β2 ) 1 m l Z ; % SD S, 2 s; 28 5 m in, 012 m l 4 m ggm l ON PG ( 2Β2D 2 ) Z, ;, 015 m l 1 m o lgl N a2co 3, ; rgm in 10 m in, A 420nm Β2 : 1000 A 420g[ ( ) (,m in) ] 3, 2 FM R 1 17, , 20 m RNA R T 2PCR 11 m R 2 [ 1, 2 NA,W estern 4 5 ] FM R 1, [ 11 FM R 1 12 ] 12 FM R P [ 6 ] FM R P FXR 1, FM R (F ig 1) 6 [ 8 ], FXR 1, FM R P FXR 1 FM R P lexa DNA, FXR 1 V P16 FM R P FXR 1, lexa V P16, h is lacz, FM R P FXR 1 399

5 [ 12 ], iso1 iso7, FXR 1 h is, (Ycg2trp, 2leu, 2h is, 2u ra, 2lys) (F ig 3) lacz (F ig 4) iso4 iso6 iso10 iso12 FXR 1, iso1 iso7 FXR 1 6 (F ig 1), iso1 iso7 [ 9 14, 4 14 ] iso1glexa iso7glexa, FXR 1 iso1 1 iso [ 9, CO S ] 2 FXR 1, h is lacz (F ig 3, F ig 4) F ig 3 Grow th status of yeasts bearing bo th FXR 1 and different FM RP isofo rm s on Ycg2trp, 2leu, 2h is, 2ura, 2lys C lones on Ycg2trp, 2leu, 2ura w ere streaked on Ycg2trp, 2 leu, 2h is, 2ura, 2lys p late Incubation w as at 30 fo r 72 h Iso 1 and iso 7 did no t grow F ig 4 Interactions of different FM RP isofo rm s w ith FXR 1 quantitative m easurem ent of Β2galacto si2 dase activities x θ : average of th ree m easurem ents; SD: standard devi2 ation FM R P 6 FXR FM R P FXR 1 FM R P FXR 1 Β2 (F ig 4), : iso4 iso10> iso6 iso12> iso1 1 iso7 1 FM R P, FM R P 12 N [ 13 (globu lar dom ain s), C ] iso6 iso12 C [ 14, ] C, FM R P FXR 1 C, FM R P FXR 1 F ig 4, C pn I FXR 1 FM R P FXR 1 FM R P C FXR 1 400

6 References 1 V erkerk A, Graaff E, Boulle K, E ich ier E, Koneck id, Reyniers E,M anca A, Poustka A,W illem s P, N elson D, Oo stra B A lternative sp licing in the fragile X gene FM R 1 H um M ol Genet, 1993, 2 (4) : V erheij C, Bakker C, Graaff E, Keulem ans J,W illem en R, V erkerk A, Gaijaard H, Reuser A, Hoogeveen A, Oo stra B Characterization and localization of the FM R 1 gene p roduct associated w ith fragile X syndrom e N atu re, 1993, 363: Eberhart D,M alter H, Feng Y,W arren S T he fragile X m ental retardation p ro tein is a ribonucleop ro tein containing bo th nuclear localization and nuclear expo rt signals H um M ol Genet, 1996, 8 (8) : Khandjian E, Co rbin F,W oerly S, Rousseau F, T he fragile X m ental retardatin p ro tein is associated w ith ribo som es N a2 tu re Genetics, 1996, 12: Zhang Y, O conno r J, Siom im, Srinivasan S, D utra A, N ussbaum R, D reyfuss G T he fragile X m ental retardation syn2 drom e p ro tein interacts w ith novel homo logs FXR 1 and FXR 2 EM B O J, 1995, 14 (21) : Siom im, Zhang Y, Siom i H, D reyfuss G Specific sequences in the fragile X syndrom e p ro tein FM R 1 and the FXR p ro2 teins m ediate their binding to 60S ribo som al subunits and the interaction among them M ol Cell B iol, 1996, 16 (7) : V o jtek A, Ho llenberg S, Cooper J M amm alian Ras interacts directly w ith serinegth reonine k inase Raf Cell, 1993, 74: Sittler A, D evys D,W eber C,M andel J2L A lternative sp licing of exon 14 determ ines nuclear o r cytop lasm ic localization of fm rl p ro tein isofo rm s H um M ol Genet, 1996, 5 (1) : Bardoni B, Sittler A, Shen Y,M andel J2L N eu robiology of D iseases, ( in p ress) 10 A usubel F, B rent R, Kingston R,M oo re D, Seidm an J, Sm ith J Struh l K eds S hort P rotocols in M olecu lar B iology, 3rd ed, John W iley & Son Inc, 1992: ,,,,,, 1FM R 1 1 : 1 : (H uang Tao, L il an2ying, Fna Yu, Q in Xue2B in, Pang Zhi2L ing, Shen Yan,W u Guan2Yun Study of alternative sp licing exp ression of FM R 1 gene ln: D ep t Scientific Research, PUM C and CAM S ed A nnual R esearch M eeting of PUM C and CA M S Beijing: U nited P ress of PUM C and CAM S), 1996: E sto jak J, B rent R, Go lem is E Co rrelation of two2hybrid affinity w ith in v itro m easurem ents M ol Cell B iol, 1995, 15 (10) : Gibson T, R ice P, T homp son J, H eringa J KH dom ains w ith in the FM R 1 sequence suggest that fragile X syndrom e stem s from a defect in RNA m etabo lism T IB S, 1993, 18 (9) : A sh ley C, Sutcliffe J, Kunst C, L einr H, E ich ler E, N elson D,W arren S H um an and m urine FM R 1: alternative sp licing and translational initiation dow nstream of the CGG2repeats N atu re Genetics, 1993, 4: