Overexpression of DEAD box protein pmss116 promotes ATP-dependent splicing of a yeast group intron in vitro

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1 Nulei Aids Reserh, 1995, Vol. 23, No. 15 Overexpression of DEAD ox protein pmss116 promotes ATP-dependent spliing of yest group intron in vitro sell Niemer, Crlo Shmelzer nd G. Vlentin Borner* nstitut fur Genetik und Mikroiologie, Universitt Miinhen, Mri-Wrd-Strsse 1, D Munhen, Germny Reeived April 1, 1995; Revised nd Aepted June 19, 1995 ABSTRACT The group intron l1, the first intron of the mltohondril ytohrome gene in yest is self-spliing n vitro. Geneti evidene suggests tht frns-ting ftors re required for in vivo spliing of this intron. n ordne with these findings, we present in vitro dt showing tht spliing of h under physiologil onditions depends upon the presene of proteins of mitohondril lyste. ATP is n essentil omponent in this retion. Overexpression of the nulerenoded DEAD ox protein pmss116 results in mrked inrese in the ATP-dependent spliing tivity of the extrt, suggesting tht pmss116 my ply n importnt role in spliing of l1. NTRODUCTON n orgnisms with split genes the introns re removed from the primry trnsript y RNA spliing. Spliing of nuler premrna introns tkes ple on the splieosome, omplex ontining numerous proteins nd five smll nuler rionuleoprotein prtiles (snrnps). Notly, severl of the proteins prtiipting in splieosome ssemly or the two trnsesterifitions require ATP hydrolysis. Yet, for the hemil retion itself energy input is dispensile, s group introns undergo self-spliing without ATP (reviewed in 1). n ontrst, protein-independent utotlyti spliing of some group nd group introns hs een shown in vitro (2-5). Two lines of evidene, however, hve led to the view tht speifi fr/w-ting ftors, presumly proteins, re essentil for in vivo spliing of most, if not ll, group nd group introns (reviewed in 6). First, self-spliing n only proeed under reltively non-physiologil onditions, e.g. 60 mm Mg 2+ nd 45 C (2,5). Seond, geneti nlysis of spliing in fungl mitohondri hs resulted in the hrteriztion of numerous nuler s well s orgnellr frnj-ting mutnts tht impir spliing of one or more mitohondril introns. Two lsses of proteins tht prtiipte in spliing of orgnellr introns hve een identified y their different pttern of inheritene. Mturses re enoded y open reding frmes loted in the intron tht they help to exise. Thus their synthesis is dependent upon mitohondril trnsltion (6-9). A seond lss of muttions tht ffets spliing of orgnellr introns is loted on nuler genes whose produts re presumly imported into the mitohondri where they ssist spliing. These genes inlude, for exmple, CBP2, MSS18 nd MSS116 in yest or yt-18 in Neurospor (10-13). The nuler yest gene MSS116 ws initilly isolted vi geneti sreenrevelingtht this gene n omplement nuler mutnt defetive in spliing of severl group nd possily group introns (12). nterestingly, the sequene of MSSJ16 shows remrkle degree of sequene homology with new fmily of proteins, the DEAD ox proteins, so lled euse they shre the highly onserved motif Asp-Glu Al-Asp, together with six other onserved elements (14). Memers of this nd the relted DEAH sugroup prtiipte in vriety of RNA-ssoited funtions, e.g. initition of trnsltion, splieosoml spliing nd riosome ssemly (15-19). Some memers of the DEAD ox fmily hve een shown to possess n ATP-dependent RNA unwinding tivitiy (20-22). Most of the orgnellr spliing ftors hrterized to dte re essentil for exision of only one or of few introns nd exhiit no ovious sequene homologies mongst eh other. This speifiity of ftors for their respetive introns distinguishes orgnellr from splieosoml spliing, where roughly the sme set of ftors proess the mjority of mrnas. Consequently, it is widely elieved tht ll introns with onserved seondry strutures were originlly self-spliing. Aording to this hypothesis, it ws only lter in evolution tht protein-ssisted spliing developed independently for eh of these introns (6). Although numerous trns-ting ftors ffeting spliing of group nd group introns hve een defined y muttions in mitohondril systems, iohemil evidene for suh prtiiption is still sre nd hs een suessfully demonstrted only for some proteins involved in group intron spliing. One exmple is the CYT-18 protein in Neurospor, whih is identil to mitohondril tyrosyl-trna synthetse. The purified protein CYT-18 hs een shown to filitte spliing of the mitohondril lrge rrna intron (13,23). We were interested in identifying the trns-ting ftors tht promote spliing of group intron l 1, the first intron of the ytohrome gene in yest mitohondri. The development of n ssy omprising proteins of mitohondril lyste mde it possile to demonstrte protein-ssisted in vitro spliing of group intron. This retion is ATP-dependent. One protein involved in spliing of ll is enoded y the nuler gene *To whom orrespondene should e ddressed t present ddress: Zoologishes nstitut, UrriversitSt MUnhen, Luisenstrsse 14, D Miinhen, Germny

2 Nulei Aids Reserh, 1995, Vol. 23, No MSS116. Overexpression of this gene inreses the ATP-dependent spliing tivity of the extrt Prmeters of pmss116-promoted spliing of l 1 hve een hrterized. MATERALS AND METHODS Strins of Shromyes erevisie, growth onditions nd preprtion of mitohondri! mtrix proteins The wild-type strin used in this study ws S.erevisie A237 (MAT, trpl, ur3-52, rho + ), whih is devoid of mitohondril DNse nd RNse NUC1 (24). The host strin for trnsformtions ws A237, onstruting A237/pGU (ontining plsmid pgu) nd the AfSS//6-overexpressing strin A237/pGU:MSS116 (ontining MSS116 in plsmid pgu). Cultures of A237 were grown t 30 C in YP medium supplemented with 3% glyerol. Strins ontining the pgu plsmid or derivtive thereof were grown in miniml medium supplemented with essentil mino ids t 30 C. For preprtion of mitohondril mtrix proteins ells were grown to log phse nd hrvested t titer of 10 7 ells/ml. Mitohondri were prepred from spheroplsts y osmoti lysis nd purified y differentil entrifugtion. Mtrix proteins were otined y sonition of the orgnelles nd susequent entrifugtion t g nd g to remove mitohondril memrnes nd riosomes, respetively. The olleted extrt (S100) ws dilyzed nd stored t-70 C in 20% glyerol, 0.1 mm EDTA, 1 mm PMSF, 1 mm DTT, 20 mm HEPES-KOH, ph 7.4, 100 mm NCl. Preprtion of RNA Trnsripts were synthesized y in vitro trnsription with T3 RNA polymerse. Trnsription ssys were rried out in 20 u.1 retion ontining 5 \xg templte DNA, 40 U enzyme, 40 mm Tris-HCl, ph 7.5,6 mm MgCl 2,10 mm DTT, 4 mm spermidine, 500 nm eh rntp for 2 h t 37 C. For genertion of internlly leled trnsripts 10 u.ci [ 32 P]UTP were dded to the ssy. Following trnsription prernas were eletrophoresed on 5% polyrylmide-8 M ure gels, utordiogrphed, extrted from the gel nd purified. Templtes for synthesis of prerna were plsmid BS/l 1/5+24 (25), whih hrors the omplete intron ll, 35 nt of the 5' exon nd 238 nt of the 3' exon, nd plsmid BS/5 (26), respetively. n vitro spliing ssys n vitro self-spliing ws performed in 20 u,l Tris-HCl, ph 7.5, 60 mm MgCl 2, 2mM spermidine, 500 mm NH4C t 45 C for 15 min. The retion ws stopped y ethnol preipittion. The resulting pellet ws wshed with 70% ethnol nd dried under vuum. Protein-dependent in vitro spliing ws performed in 40 il 10 mm HEPES-KOH, ph 7.5, 10 mm Tris-HCl, ph 7.5, 100 mm NCl, 10 mm MgCl 2, 2 mm DTT, 2 mm ATP, 10 ig Esherihi oli trna nd 10 U RNse inhiitor t 28 C for vrious times. The retion ws stopped with 60 u.150 mm NA, ph 5.2, 50 mm EDTA, 0.1% SDS, 2.5 u.1 proteinse K (20 mg/ml). After phenol/hloroform extrtion the retion produts were preipitted with ethnol nd the resulting pellet wshed nd dried. The ssys were done in the presene of u.g mitohondri] mtrix proteins nd in the presene of extrts previously digested with proteinse Krespetively.The produts of in vitro spliing retions were seprted y eletrophoresis on denturing 5% polyrylmide gels. Constrution of plsmid pgv:mss116 Plsmid CA7 (27) ontins the omplete sequene of MSS116 with n dditionl 5' 600 nt nd 3' 350 nt s genomi HindB frgment. The Hindlll frgment of CA7 ws loned into Bluesript SK (Strtgene). The 5' non-oding region of the MSS116 sequene ws then shortened to 60 nt yrestritionof the plsmid with Bll nd Sml. Restrition of the resulting plsmid with BmH nd Sil yielded frgment ontining the MSS116 gene with 5' nd 3' non-oding regions nd djent Bluesript polylinker sequenes. This frgment ws loned into the multiopy yest 2(i plsmid pgu nd the resulting plsmid designted pgv:mss116. pgu ws derived from plsmid pgl (28) y repling the TRP1 mrker gene with the URA3 mrker gene derived from puc19. Trnsformtion of A237 ws rried out ording to Klee et l. (29). Trnsformnts were seleted on syntheti omplete medium without uril. soltion of S.erevisie RNA nd Northern nlysis Cells were hrvested in log phse, wshed one in H 2 O nd the pellet frozen in liquid nitrogen. The pellet ws resuspended in extrtion uffer (0.15 M NCl, 50 mm Tris-HCl, ph 7.5,5 mm EDTA, 5% SDS). Cells were roken y vortexing with glss eds nd phenol/hloroform for 5 min. Nulei ids were extrted three times with phenol/hloroform nd preipitted from the liquid phse with ethnol, 0.3 M NA. nution for 3 h in 10 mm Tris-HCl, ph 8.0, 1 mm EDTA, 3 M LiCl preipitted the RNA. Whole-ell RNA ws seprted on formldehyde-grose gels nd trnsferred to nylon memrnes (Amershm). DNA proes were 32 P-leled y nik trnsltion (30). The nik-trnslted hyridiztion proe for detetion of MSS116 sequenes ws the desried HindlW frgment of plsmid CA7 (27). Anlysis of pmss116 protein Rit ntiodies were rised ginst 14 mino id domin derived from mino id positions of pmss 116 (sequene S-R-P-R-T-R-S-R-E-D-D-D-E-V). SDS-PAGE ws rried out y the method of Lemmli (31), using 5% stking gel nd 12% seprting gel. Eh lne ws loded with 30 Xg mitohondril proteins. After trnsferring the seprted proteins to n mmoilon P memrne (Millipore) the produts ould e proed with ntiserum P, direted ginst the ove-desried mino id domin. mmunolots were stined with ECL (Amershm). RESULTS Protein-dependent spliing of ll depends on ATP The group intron ll hs previously een shown to undergo self-spliing in vitro, retion identil with splieosoml spliing in mehnism,resultingin the exision of n intron lrit vi two susequent trnsesterifitions (5). Yet effiient selfspliing of ll requires high slt onentrtions nd high temperture, suggesting tht /r/w-ting ftors re essentil for the in vivo exision of this intron. n order to hrterize suh ftors, we estlished n in vitro system whih llowed us to ssy for protein-dependent spliing.

3 2968 Nulei Aids Reserh, 1995, Vol. 23, No min M 30min 60 min 120 min mmatp M L-3'E _ L «P 5E-3E ^. Figure 2. ATP dependene of spliing of l 1. Spliing ssys in the presene of ntive mitohondril proteins were erned out s desried (see Mterils nd Methods) with inresing onentrtions of ATP (0-10 mm). Figure 1. Protein-dependent spliing of ll in the presene of mitohondril SlOO extrt 32P-Leled l/5+24 RNA ('prerna') ws inuted for vrious times in the presene of 15 ig mitohondri] mtrix proteins pretreted with proteinse K (), 15 ig ntive proteins () nd in the sene of proteins (). The produts were gel eletrophoresed on 5% denturing polyrylmide gels. Lne M shows the produts of n utotjyti retion of prerna. L-3'E, lrit with ovlenuy ound 3' exon; L, lrit; P, prerna; 1, liner intron, 5'E-3'E, ligted exons. An internlly 32P-leled trnsript hroring l 1 ws inuted under vrious onditions with n SlOO extrt prepred from rude mitohondril lyste. To minimize non-speifi endogeneous nulese tivity, mitohondri were isolted from yest strin A237, whih is defiient in the extremely tive mitohondri] NUCl exo-endonulese (24). After inution of the trnsript with the mitohondril SlOO extrt under onditions resemling the physiologil stte nd in the presene of ATP the RNA ws nlyzed y polyrylmide gel eletrophoresis. To monitor the effiieny of spliing we sreened for the presene of n intron lrit, whih is esy to detet due to its low eletrophoreti moility. As n e inferred from the time ourse shown in Figure 1, spliing under physiologil onditions only proeeded in the presene of mitohondril SlOO extrt (lnes ), wheres no lrit ws formed if the RNA ws inuted in retion uffer lone (lnes ). Preinution with proteinse K resulted in mrked redution in spliing tivity, inditing the prtiiption of proteins in the retion (lnes ). The time ourse shows tht spliing inreses linerly for 2 h nd lrit formtion plteus t ~5% of the input prerna (Fig. 1). n omprison with optimized in vitro self-spliing, where >50% of the intron ws exised from the preursor fter 30 min (see lne M in Fig. 1), the protein-dependent in vitro retion is reltively slow. Lyste-dependent spliing of group intron ll n only proeed in the presene of ATP nd Mg 2+. Aording to the experiment shown in Figure 2, there is n optimum spliing tivity t 2 mm ATP. This tivity drops t higher ATP onentrtions, however, when the Mg2+ onentrtion is inresed onomitntly with ATP, protein-dependent spliing tivity remins unhnged (not shown). The pprent deline in spliing tivity t higher ATP onentrtions thus seems to e due to the omplexing of ATP with Mg 2+, rther thn to n inhiitory effet of high ATP onentrtions. As Mg 2+ onentrtions >10 mm lso promote utotlyti spliing, ll susequent experiments were rried out t 10 mm Mg 2+ nd 2 mm ATP. The ATP dependene of the spliing tivity oserved in these experiments with mitohondril lystes lerly distinguishes protein-dependent spliing of ll from utotlyti spliing of group introns, pthwy tht hs een shown to e intrinsilly independent of nuleotide o-ftor (3-5). Overexpression of MSS116 Hving estlished tht protein-ssisted spliing of l 1 depends on ATP, we further investigted the nture of this spliing tivity. As shown in lnes of Figure 1, preinution of the extrt with proteinse K resulted in ler redution in spliing tivity. The fint nd in lnes fter 60 nd 120 min respetively ould e due to inomplete digestion of lyste proteins y proteinse K. Another possile explntion ould e tht proteinse K-generted peptide frgments my hve ertin stilizing effet on the tlytilly ompetent onformtion of this group intron. Peptides with high positive hrge hve previously een shown to stimulte tlysis of the hmmerhed riozyme (40). n ontrst, spliing tivity proved insensitive to mirool nulese nd thus seems to onsist of one or severl protein omponent(s) lking n essentil RNA omponent (not shown). At the time of our initil experiments it ws shown tht the yest gene MSS116 n omplement nuler mutnt defiient in spliing of l 1, nother group intron nd possily severl group introns in yest mitohondri (12). n the sme study the gene MSS116 ws shown to e loted on 2.9 k genomi Hin&\\\ frgment with n ORF oding for protein with 664 mino ids.

4 Nulei Aids Reserh, 1995, Vol. 23, No The derived protein sequene of pmss 116 shres remrkle homology with memers of the DEAD/H ox fmily, protein fmily some memers of whih hve demonstrted ATPpGU MSS pgu MSS dependent RNA unwinding tivity (14,20-22). Compred with the prototypes of this fmily, p68 nd ef4, pmssl 16 possesses 97,4 n dditionl streth of 30 minly positively hrged mino ids t its N-teiminus, s expeted of presequene required for 4.4 trgeting proteins into mitohondri (12,32). 72 kd 69 We inferred tht the gene produt of MSS116 ould e possile MSS116 ndidte for the ATP-dependent spliing tivity oserved in 2,4 mitohondril lystes. Therefore, we hve ompred the spliing 2.1 kb tivity of n extrt from n MS577<5-overexpressing strin with tht of strin rrying the hromosoml opy of this gene only MSS116 ws overexpressed under trnsriptionl ontrol of the onstitutive GPD promotor (33). To remove possile endogeneous expression signls, the sequene 5' of the MSS116 initition odon ws shortened to length of 60 nt The resulting 2.3 k frgment C ontining MSS116 flnked y some non-oding sequenes ws inserted into the 2u. plsmid pgu ehind the GPD promotor. Vetor pgu MSS K pgu is derived from pgl (28) y repling the seletion mrker t TRP1 with URA3. Susequendy the onstrut pg\j:mss116 ws trnsformed into yest strin A237. As negtive ontrol we used extrts from wild-type strin A237, whih ontins single opy of MSS116 ompred with the overexpressing strin. To ensure 97,4 similr growth onditions A237 ws trnsformed with plsmid pgu lking the MSSU6 insert (A237/pGU). -72kD Anlysis of the expression levels of MSS116 ws performed y 69 MSS116 Northern nd Western lots of A237/pGU:MS5776 nd A237/pGU respetively. A RNA lot proed with n A/SS776~-speifi DNA frgment showed signl of 2.1 k tht ws present in A237/pGU:MSSJ16 t high onentrtion, while the trnsript from the hromosoml gene ws hrdly detetle 46 in A237/pGU (Fig. 3). Thus MSS116 is trnsried with high effiieny into stle mrna in the overexpressing strin. As next step n ntiody ws rised ginst the 14mer peptide P Figure 3. Overexpression of MSS 116 in yest strin trnsformed with plsmid (S-R-P-R-T-R-S-R-E-D-D-D-E-V), representing n N-terminl pgumss116. () Northern nlysis of whole-ell RNA from A237/pGU pmss 116 epitope (mino id positions 67-80) of potentilly (pglf) ompred with RNA from A237/pGU:W55//6 (MSS). The RNA ws seprted on formldehyde grose gel, trnsferred to nylon memrne nd high immunodominne s predited y the progrm DNA STAR proed with nik-trnslted M55//6-speifi DNA frgment. The hyridiz(34,35). The nti-pi serum, ut not pre-immune serum, reogtion proe detets 2.1 k trnsript, speifilly overexpressed in nized protein in the mitohondril lyste with n pprent A237/pGU:MSS//6. () mmunolot nlysis of mitohondril proteins. moleulr weight of 72 kd. This oinides with the size Mitohondri] mtrix proteins from the strins indited were seprted on 12% SDS polyrylmide gels with 5% slking gel. n the immunolot shown predited for pmss 116 lking the ~30 mino ids of puttive nti-pmssl 16 ntiserum P ws used for deortion. The moleulr weight of import sequene t its N-terminus (Fig. 3). While the silver pmss16 ws estimted from omprison with stndrd protein mrker, stined SDS-PAGE gel showed no differene in the expression indited on the left side of the lot () mmunolot nlysis of proteins shows pttern of mitohondri! proteins etween A237/pGU.MSSl16 the loliztion of pmss 116 in the mitohondril mtrix. dentil mounts of nd A237/pGU (not shown), the Western lot in Figure 3 proteins (20 ig) were seprted y SDS-PAGE nd deorted ording to (). Mtrix proteins re from A237/pGU (pgu) nd A237/pGU:MSSl 16 (MSS). reveled tht the onentrtion of protein pmss116 is t lest Lne K shows mitohondril memrne proteins from A237/pGU:MSS 116 for 30-fold higher in A237/pGU:AfSS776 thn in A237/pGU. The omprison. ellulr loliztion of pmss116 ws determined y immunodeortion of the mitohondril mtrix frtion, s ompred with the memrne frtion. Anti-Pi only reognized nd in Figure 4, the level of lrit formtion ws signifintly inresed the mtrix frtion. This experiment onfirmed umultion of in the A231/pGV:MSS116 extrt (lnes ) ompred with the pmss116 in the mitohondril mtrix (Fig. 3). A237/pGU extrt (lnes ). The differene in spliing tivities is espeilly pprent fter 60 min nd orreltes roughly with the level of overexpression oserved in the Western lot. Aording n vitro spliing of ll is promoted y overexpression of to the experiment shown in Figure 4, lrit formtion seems to MSS116 hve lg period in the lyste from the overexpressing strin. However, this oservtion ws not onsistent nd ws not n order to investigte whether the level of MSS116 expression investigted further. We lso oserved tht extrts from strin orreltes with the spliing tivity found in mitohondri! A237/pGU exhiited redued spliing tivity thn those from lystes we inuted l 1/8+24 prerna (25) with the two non-trnsformed A237. This is proly due to the differene in respetive extrts. As n e seen in the time ourse shown in

5 2970 Nulei Aids Reserh, 1995, Vol. 23, No min 30 min Control 60 min M G A M L Figure 4. Enhned spliing tivity of extrts derived from A237/pGU:AfSS//6 ompred with A237/pGU. Spliing ssys were performed s desried (see Mterils nd Methods) in the sene of proteins (), in the presene of mitohondri] proteins from A237/pGU () nd in the presene of mitohondril proteins from A237/pGU:MS5//6 (). pgu U C G A U C MSS r G A U C - i Figure 5. The spliing retion speifilly requires ATP. Spliing ssys were rried out in the sene of proteins (ontrol), with A237/pGU extrts (pgu) nd with protein extrts from A237/pGU:A/SS//<5 (MSS). Where indited, 2 mm ATP in the retion uffer ws sustituted y 2 mm GTP (G), CTP (C) or UTP (U) respetively. nution ws performed for 45 min. DSCUSSON growth medium (A237/pGU ws grown on seletive miniml medium). n prllel preprtions from A237/pGU nd A237/pGU:M55776, however, extrts from A237/pGU:A/SS7/6~ lwys exhiited higher spliing tivity thn A237/pGU. Our results thus demonstrte tht overexpression of nuler gene MSS 116 lone is suffiient to inrese ATP-dependent spliing of group intron l 1 in mitohondri] lyste. n order to eluidte this proess further, the speifiity of pmss 116-promoted spliing with respet to o-ftors nd the RNA sustrte ws investigted. Aording to the results shown in Figure 5, pmssl 16-promoted spliing of ll proeeds only in the presene of ATP, while none of the other stndrd rntps n e used. A similir speifiity for ATP hs lso een shown for other DEAD ox porteins, inluding DpA (19) nd ef4 (21). To lern more out the sustrte speifiity of pmssl 16promoted spliing we tested whether A231/pG\J:MSS116 lyste n lso enhne spliing of other group introns. ntron 5, the lst intron of the oxl gene in yest mitohondri is losely relted to l 1 in seondry struture nd primry sequene, oth introns elonging to the sme sugroup of group introns (36). n vitro self-spliing of!5 hs een oserved under onditions similir to those of l 1 (3,4). However, neither the extrt from A237/pGU nor tht from A237/pGU:A/55/76 hd ny detetle effet on spliing of 5, i.e. no lrit formtion ould e oserved under onditions optimized for ll spliing (not shown). Our findings re onsistent with results from the geneti sreen, showing tht MSS116 is not essentil for in vivo exision of 5 (12). The oservtion tht overexpression of MSS 116 is not suffiient to generlly enhne spliing of group introns seems to indite tht pmss 116 does not ffet l 1 spliing s sequene-non-speifi RNA helise, n tivity oserved for some DEAD ox proteins (20-22). n this work we hve nlyzed protein-dependent spliing of mitohondril yest intron ll, n in vitro utotlyti group intron. We estlished n in vitro system tht llows spliing of l 1 prerna only in the presene of mitohondril S100 extrt ATP is n essentil omponent in this retion. The ft tht the spliing tivity of the extrt is signifintly inresed y overexpression of DEAD ox protein pmss 116 suggests tht this protein my ply n importnt role in spliing of l 1. n vitro spliing of ll under physiologil onditions depends on protein lyste nd ATP Spliing of group introns proeeds vi two trnsesterifitions, resulting in the exision of rnhed struture, the intron lrit (3-5). Extensive reserh with mutnts of utotlyti group introns hs provided reltively ler onept of the funtion of onserved seondry struture domins 1-6 for the spliing retion (36,37). These dt imply tht ll tlyti tivities required for spliing re inherent in the onserved struture of the intron RNA. However, t physiologil Mg 2+ onentrtions nd t low temperture no utotlyti tivity ould e oserved. Our results show tht under these onditions effiient lrit formtion (nd onsequently formtion of the funtionl mrna) depends upon the presene of one or severl protein(s) from mitohondril S100 extrt. This finding supports the hypothesis tht the utotlyti retion, whih is hrdly detetle under physiologil in vitro onditions, n e enhned onsiderly y the tion of trns-ting ftors. The oservtion tht proteinse K, ut not mirool nulese, redues spliing nd tht ATP is n essentil oftor, whih nnot e sustituted y other rionuleotide triphosphtes, suggests tht DEAD ox protein pmss 116 ould e the ndidte protein. The speifiity for ATP is mjor hrteristi of the DEAD ox proteins investigted

6 Nulei Aids Reserh, 1995, Vol. 23, No to dte (19-21). The dependene on ATP distinguishes the protein-dependent pthwy from in vitro self-spliing of group introns, whih does not require n externl energy soure. n oth the protein-dependent nd the utotlyti pthwys the intron is exised s lrit. t therefore seems resonle to ssume tht protein-dependent nd utotlyti spliing of ll proeed vi the sme mehnism, despite the ft tht protein-dependent spliing relies on the presene of ATP. The role of pmss116 in spliing of ll A geneti pproh hs previously shown tht ll nnot e exised from the primry trnsript in yest strins rrying muttion in the nuler gene MSSJ16(\2). We hve onstruted strin tht overexpresses DEAD ox protein pmss116. Northern nlysis shows tht this gene is trnsried in strin A237/pG\J:MSS116 with onsiderle effiieny into stle mrna. t seems, therefore, tht the low undne of MSS116 mrna in the wild-type is due to low level of trnsription, rther thn to the instility of the trnsript, s disussed erlier (27). The trnsltion produt pmss116 is present in the mitohondril mtrix of strin A237/pGU:A/55//6 s solule ompound t onsiderly inresed onentrtion s ompred with the wild-type strin. Dt from our in vitro spliing ssy show tht overexpression of MSS116 signifintly enhnes protein-dependent spliing of l 1. As in the wild-type lyste, the retion is dependent on ATP. A plusile interprettion of these results is tht the ATPdependent spliing tivity n e ttriuted to DEAD ox protein pmss116. This is supported y t lest two lines of evidene. First, the spliing tivity present in mitohondril extrts exhiits ll the iohemil hrteristis oserved for DEAD ox proteins, nmely dependene on ATP nd Mg 2+ (17-20). Seondly, Western nlysis hs provided evidene tht overexpressed pmss116 is umulted in the mitohondri] mtrix, s lredy suggested y die puttive import sequene t the N-terminus of the protein (12). An indiret influene of pmss116 on ll spliing vi mitohondril trnsltion n e exluded, sine spliing of l 1 does not require mitohondrilly enoded mturse (38). Our results, of ourse, do not prelude the possiility tht pmss116 ould possily e ytoplsmi trnsltion ftor tht indues the synthesis of spliing ftor speifi for l 1 nd other mitohondril introns. Thus we would like to emphsize tht onlusive eluidtion of the role of pmss116 in spliing of ll will require purifition of this protein. Addition of the purified omponent to the mitohondril lyste should promote spliing of l 1 similrly to the overexpression desried in this work. A model for the intertion etween pmss116 nd ll Apprently, fter trnsription only ertin suset of prerna moleules hs the proper tertiry struture required for RNA tlyzed spliing. t hs een speulted tht severl yles of unfolding nd refolding promoted y n RNA helise tivity ould inrese the proportion of retive prerna moleules nd therey the effiieny of the overll retion. A previous model suggested tht pmss 116 might promote spliing of, t lest, three mitohondril group nd group introns vi suh n RNA unwinding tivity (12). Unwinding of syntheti RNA sustrtes hs een oserved for severl DEAD ox proteins (20-22). However, no inresed RNA helise tivity ould e deteted in extrt A231/pGU:MSS116 s ompred with A237/pGU using onditions under whih p68 unwinds smll syntheti RNA moleules (dt not shown). t thus seems unlikely tht pmss 116 hs sequene-non-speifi RNA unwinding tivity. Further exmintion of the model inluded testing die effet of MSS116 overexpression on in vitro spliing of group intron 5. This intron is losely relted to ll nd requires lmost identil onditions for self-spliing. Yet we ould not detet ny effet of pmss116 overexpression on in vitro spliing of 5. This result, whih is onsistent with the geneti dt (12), my provide some insight into the mehnism of pmss 116-dependent spliing. Although group F introns ll nd 5 hve very similir seondry (nd mye tertiry) struture, this ommon feture is oviously not suffiient for intertion with pmss116, s n e onluded from our experimentl results. One rther unlikely explntion ould e tht pmss116 ts s n RNA helise speifi for the unwinding of n unknown sudomin of l 1. Suh primry trget would hve to e ommon to ll group nd group introns whih nnot splie in n MSS116 mutnt (12), ut sent from 5. Thus more plusile model would e tht pmss 116 n only intert with its RNA sustrte(s) due to essory ftors speifi for therespetiveintrons. This would e onsistent with the hypothesis of n independent development of protein-ssisted spliing in different group introns. Evolutionry implitions Spliing of group F introns nd splieosoml spliing proeed vi the sme moleulr mehnism. This oservtion hs prompted speultion tht group introns my e the evolutionry nestors of splieosoml introns. Aording to this model, splieosoml introns grdully developed from group introns y: (i) eoming inresingly dependent upon trns-txng protein ftors; (ii) trnsposition of severl formerly w-ting RNA strutures into fr/u-ting snrnas (39). Until now no similrities with respet to trns-ting ftors hve een desried. Our results show tht spliing of group intron depends on ATP nd t lest one protein elonging to the DEAD ox fmily. 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