ATP dependent i// yilro release of labeled I:N4 from nuc,lei has been studied in HeLa cells. The release of label as TCA insoluble and soluble radioactivity from nuc*lri was dependent, on temperature and stimulated by ATP. Although protein was also released there was no significant cffecat of iZTP on this reacation. .4 variety of other nucleotides, pyrophosphate, and li:L)T,4, could replace ATP which srlggested that these compounds were acting by chelating magnesium ion. This view is supported bJ the findings that magnesium inhibited RNA release from HeI,a nuclei. In addition, magnesium also stimulat,ed the degradat,ion of any RNA that was rclcased from the nuclei. The restllt,s indicate that the ,4TP dependent release of l(NA from nllrlei, which has been reported in several systems, may be due to the ability of .4TP to chelate magnesium, an inhibitor of I:NA release.
Most nuclear RKA is hcterogcncous, has DNA-like base composition (l-3), and has been found as large molecules of spwific ribonucleoprotein complexw (4, 5). A part of this complex is transported to t#hc cyt’oplasm and is incorporated as messenger RKA (mRNA) into polysomcs (G-S). The nature of mRNA in the cytoplasm of eukaryot’ic cells (9-14) and its in r&o t,ransport from nuclei to cytoplasm have been studied in rat liver nuclei (15-20) and in adenovirus-2 infcctcd ICI3 ccl1 nuclei (21, 22). Init,iallJ Schneider (15) and more rcccntly Ishika\v:t et al. (16) have examined this process in considcrablc dct,ail. Thcsc aut#hors haw found t,hat Rn’A release from liver nuclei is dependent on temperat’urc and st,imulat cd by ATI’ but inhibited by l\‘Ig?T (15, 16, 21). Despite the considerable amount of work in this area t,hc role of ATI’ and ot,hcr nucloo~ tides in this process is still unclear. In the present report \ve have studied th(x in vitro rrlrsse of RNA and protc+n from HcLa nuclei and have cxamincd the rol(> of ATI’ and 31g3+ in this procws. MATIWIALS
AhIPPCP, 1 Abbreviations: AMP; GMPPCP, @-7.met,hylene t,riclrloroac~eti(, acid.
(‘hemicals. 15.“Hluridine (a9 Ci/mmole) and [U-‘“Clprotein hydrolysate (5i mCi/mole-atom carbon) were purchased from Amersham/Pe:trlr. 160 Copyright -411 rights
0 1973 hy Academic Press, of reproduction in any form
r‘-“C]thymidine (‘i.5 mCi/mmole) and [:qH]-IJTP (15 Ci/mmole) were purc*hasrd from Cal ;2tomics and Schwartz ISiochemicals, respectively. 8-rhIethylene ATP (AMPPCP)’ was purchased from Miles Laboratories and ( :1IPPCP was obtained from 1)r. I). RIiller of the ltoche Institute. Other chemicals were of reagent grade. Culture oj" cells, prepuratiou oj ticrrlri, und 106,OOOg .srcpernatto/ 1 (c~~loso~). I%eLa 8-z and mouse L-929 cells were grown in sllspension in Joklik modified minimum essential rncdilun (hIEM, Grand Island Biological) supplemented with 5C; fetal calf strum. All thr nuclei Itsed in the present. studies COW taincd labeled proteins and l
Herkowitz et al. (23). Washed cells were suspended in 40 ml solution of 1 rn~ potassium phosphate (pH i.0) and 10 nnr NaCl, and nftcr 30 min at O”(1, disrnptrd in a I)ounce homogenizer with lo-12 strokes of the pestle. The homogenate was centrifuged at 3OGOgfor 5 min; the pellet was resuspended in 10 nrl of a sollrtion of 1 ITIM potassium phosphate (~1% i.O), 320 mu sucrose, 1 rnRI RlgCly ! and 0.37; Triton N-101, subjected to 10 strokes 111t,hc homogenizer and centrifuged as before. After two additional Triton washes, the pellet was washed once with 10 ml of 250 mu sucrose and suspended in a small volume (S-10 ml) of 250 rnM sucrose. Protein determination (24) was made on the nuclear SUSpension in 2 M NaCl. The protein concentration of the final nIlclear suspension was about 5 mg/‘ml. .\n aliquot of the nuclear suspension was assayed for 57, cold trichloroacctic acid (TCA) insohlble rndioactivitv by filtering the TCA extract through a Rlillipore filter, and dissolving the filter in a scintillation fluid (25). An aliquot of the suspension was also dissolved in the colmting fluid withotlt filtering to dcterniinr the total radioactivity (‘U’A-insoluble and -soluble counts). All radionct ivr measurements were done with a Beckman IS250 scintillation colmtcr. The nuclear susperlsion was stored in small aliquots in liquid nitrogr’n To prepare nuclei containing [“C]t,hymidirle-labrlrd l)?jA, 1 X IO” cells were suspended in IjZBS :tt 37°C and labeled with 0.5 ml of [l*C]thymidinc (50 ~Ci/ml) for 50 min at, 37°C. To prepare unlabeled nuclei, 1.5 X lo9 cells were iiaed. Suclci were prepared following the proc*edurc d(lscribed. In l,ilro assrct~ for R.Y.1 n~id prokin release, 1j.v. I wlcnsc crud DB,1 -tzepeldet/t IL\-. 1 polynwtrse ,~~OMIIhe /~~rc/ci. The iu zlifro incubation mixture (0.5 ml) contained 50 mu Tris-TIC1 (pH 7.5j, 250 U,V s;ll,‘rose, 25 nm Kicl, 2 rn>f &mcrcapt oethanol, llllcl(li c~clrlival~~nt to 225-250 pg protein, and other ~on~ponents (RIgCl, , nuclcotidrs, etc.) listc,d in 111~legends to figTIres. After incubation, the reaction mixture was dilLItrt1 to 3 ml with the incllbaI ioll tniffer cant aining 5 ~IRI hlgCln The nuclei wrrc pelleted by centrifugation at 12oOg for 10 rnin, :cntl t 0 the slipernatant 0.15 ml lOOr TCA was added. Aliquots of 2.5 ml and 0.5 ml were assayed f’clr cold 5’;, TCA insoluble and total radioactivity, rcspc,ctivcly. I:c~l~~sc of labeled I)NA from the nuclei was assaycd in t hc above irrc~llbntion mixture at 0 and 5 ~nzr >IgCl, in the :tl)srnc*e or in thcl prrsrncc of 0, -1, :III~ 8 ,u\t ilTP. I:5 .A polymerasc~ assay was carried ollt following lhcx procedlirr of Sugdrn and S:mibrook (26). 1’111:~bc~lrti ITcLa nllclei wcrc inrubated in the i)~ 7,ilr.f~ irbc.rll,:ttion rrristllrc containiIrg 5 mM Mg(:l?
and 0, 5, or 8 nn~ ATP for 15 min at 30°C. The rraction mixture was chilled and the nuclei were removed by rent,rifugation. The sllpcrnatant) was dialyzed in standard bluffer (see 26) for J hr at 0°C before assaying for polymerase. Pol~gucrylamide gel elecirophoresis of RSLt. IlKA present in [3H]uridirle-labelcd whole nuclei or released from the nuelci after ATP incubation was estrarted by the Sl)S hot phenol method described previously (2’2). The cstracted RNA was precipit.ated by 2 M lit.hillrn chloride, rrprrcipitated 1,~. ethanol, and analyzed on 2.5’:, acryl:m~idc gels containing 0.5’/, agarose (2;). Thin layer chrotnc~/ogrtrpli!/ (f(c). .4 solvent system containing isobrltyric :Icid::Inlmorri:t:\~nter (66: 1:33) at pfI 3.7 was Ilsed to separate and identify the soluble uridinc nu(~l(~otides released from thr nrlclri. 1:15S;UI,TS .4?;1) I)ISCCSSION
4 6 ATP( mM)
FIG. 2. IXect of temperature and ATP concentration on t.he relense of RN-4 and protein from uridinc and [‘C]protein hydrolysnt,c lnbelcd IlcLa nuclei. Nuclei were incubated in the presence of 4 mx MgCln and varying concentrations of -4TP for 25 min xs described in Rlateri:tls and Methods. After incubation, nllclci were srparatcd by cent,rifugat.ion and TC&insolnble (Fig. 24) and total (Fig. 21%) “11 and l”C r:tdio:wtivit,y presents in 2.5 ml of the snpernatant was assayed as described in Mntcrials and 3Iethods. Input, radioactivity/‘incubat ion = 140,000 ,‘pn> :jIT xnd 50,000 cpm I’(‘.
sh0\~11j no effect’ of A!Tl’ on the relcuse of protein n-as olactrvcd. I+&ue 2B shows that tot al tritium relr:w was considerably higher th:lu TCA insoluble t,rit.ium wleascd seen in
;iL, , , ,_, -j 0 - 20 40
I I I A. TCA INSOLUBLE
FIG. 3. SDS polyxrrylamidc gel electrophoresis pattern of the RN.4 released from the nuclei after incubating [3H]~~ridine-lahelcd HeT,a nuclei in 7 mM 4TP and 4 rnsf MgCl? at 30°C for 15 min. The RNA, extracted from the supernntant after rcmoving nuclei by cent riflLg:ttion, was subjecbed to electrophoresis as described in >1aterials and Methods. A parallel gel was r,m wit,h total nuclear RNA extracted from whole nuclei as described in Materials and Methods. l
Whcr nwleoside di- and triphosphatw including ADP, CTl’, GTP, UTL’, the nc)~ hydrolyzable analogs AMPPCP, GMI’I’CI’, and inorganic pyrophosphate st,imulatcd RX&4 release similar to ATI’, whereas Al\ll’ and inorganic phosphate were ineffwtivct. Ishikawa et al. (16) rctportcd that ADI’ could not, replact: ATP in their system in cont,rast to the present results. The abilit,Jof AMPPCl’ to function suggested that hydrolysis of tho nuclw)tid(l \vas not involved in t)hc releaw of RNA from the nuclws. Figuw 5 sho1v-s the c8cct of RTI’ and A1II’I’CI’ wnwnt,ration on hhc r&asc: of RX.4 from [“Hluridinc-labeled H&n nu&i after incubat,ion for 15 min at 30°C in
0; TIME (MINUTES)
FIG. 4. Kinetics of ATP dependent release of ILNA from [3H]uridirle-labeled HeLa nuclei. V\lclei were incubakd in the presence of 6.5 mM ATP and 5 rnM MgCly for different periods of time at 30°C and the amollnts of TCA-insoluble (Fig. 1-4) and total (Fig. 4R) radioactivity present in 2.5 ml of the supernatant were determined. Inpllt radio:Ictivity/incllbatiorl = 50,000 cpm 311.
the prcwnw of 5 mM MgCls . In the figuw, the roloasc~ of both TCA-insoluble radioactivity and t,otal radioactivity is similar wit)h both compounds. Tn comparable cxpcrimclnts there was no significant release of protein from the nucleus as a function of either ATP or AhlPPCP concentrat8ion. The, RNA rc+aw was maximum at 6-7 rnlr nuclcotidc. The tot,al RNA released in the pxpwimc~nt,s w-as considerably highw than TCA-insoluble RKA at Cach concentration of t,h(: triphosphatc as shown abow in Figs. 2 and 1.
ATP [TCA INSOLUBLE)
FIG. 5. ISficrt of ATP and ARIPPCP ~c,nceIllrRticIn on 111~ release of J’LNA front j’:H]uridineJabelect HeLa nllclei. Nuclei wcrc inc~~bated in the prcscnw of 5 nlb~ i&$1:, for 15 rnin at, 30°C and the a~~~ourr~s of TCA-insoluhlc and total radioactivit? present in 2.5 ml of the s~~prin:ttant wcw determined. Inpllt rndioactivit~/irrc~~t~~~t ion = 52,000 cpm 31J.
I?Ic;. fi. Effect of nlagnesirun roncentration on the release of l
nucleus which might bc inhibitor>- for RNA relraac from the nucleus is bc4tig rcmovcd by t,hrx nuclcotide. In addition, cwenti:111> all of the, radioactivity rc~l~~wc~l undw these condit,ions (ATI’ mmus Ill&+) Leas insoluble in TCIA, i.c>., not dcgradcd to smnll frqmcwts. TVith thcl addition of grcwtcr th:ln
2 m&I Xg3+ thcrc is :I decrease in both the total and TCA-insoluble tritium released. Although not showy, thcrc was no eff ert of nfgp+conccnt’ration on the release of protein from t,hc nuclei. If l\Ig”+ inhibits the wlease of RNA from the nucleus as indiratjcld by t,he results in Fig. G! and nucleot8idcs such as hT1’ are stimulat,ing t.hc reaction by removing l\lg”+, it would be expwtcld that an inorganic chelator such as EDTd would also st~imulnte the release of Rn’=\. Previous st,udies have not demonstrated a stimulatory cffcct’ of EDT-1 on RlvA release from nuclei in other systems (15, 16). Figure 7 shows the effect, of different concentrations of EDTA on RNA release from HeLa nuclei in the presence of 5 ml1 -IlgCl, . It can be seen that t,he release of RNA (TCA-insoluble 3H) is stimulated lq- EDT.4; and at, low concentrations of EDTA (2.5 rnkl) a considerable amount of the released RKA is degraded to TCX-soluble radiowtivit,)-. However, at higher concentrations of EDTB (6 mar alld abow), there is littlr degradation of the RXL’,4 sinw TC&insoluble and total radioactivity wmain about, t.he same. The
kinet,ics of RNA release at 6.7 rnM IZDT&4 arc shown in Fig. 8. It’ is seen that EDTX caused a t,wofold increase in t.he rxte of release of RNA. In the absence of ;\lgCIZ , low cone&rations of EDTA released more rudioactivit\compared to t,hnt released in the prcscnce of IIgCl, (data not shown). This could be due to the chelation of cndogenous Xg”+ in the nucleus by EDTA. Thcw results support the view that XlgLT inhibits RSA release from isolated nuclei and stimulates t,he drgradation of any RNA that is rc~lcnsed. The rclcase of RNA in these experiments did not appear to be due to :m increase in the number of lysed nuclei. Figure 1C shows a phase contrast photomicrograph of HeLa nuclei incubat’cd for 15 min at 30°C in the absence of ,4TP and nlgC1, Figures 1D and E show similar photomicrographs of nuclei incubated in t.he presence of 4 mM ATP (- NlgCl,), and in t,he presence of ,4TP plus NlgClz , wspectivel!-. These incubations did not appear to increase the number of lysed nuclei or to change the size of the nuclei. Incubation of nuclei in the presence of 8 mM ikfgclz (not shown) rcduccd the size of the nuclei considerably, an effect that could be reversed by adding ATI’. A furtlwr indication that 20 r
l?ic. 7. Effect of l,:L)TA on the release of KNA from [“Hluridille-labclcd IIcLa nuclei. Nuclei were incubated in the prcsencc of 5 111~MgC12 and varying concentrations of EI1T.4 for 15 min at 30°C and the amounts of TCA-insoluble and total radioactivity present in 2.5 ml of the supernatant determined. Input. radioactivity;‘incubation = 50,000 cpm 3H.
FIG. 8. Kinetics of E:UTA dependent release of ILNA from /“II]uridine-labeled TleLa nuclei. Suclei were incubated in the presence of 6.7 m&I F:l~)Ta and 5 mu hlgCls for different periods of time at 30°C and the amount of TCA-insoluble radioactivity present in 2.5 ml of the xupernatanl determined. Input radioactivit.y/in(:llbatior~ = 50,000 cpm “H.
the: results obkrined wrr not due to lysis of the: nuclei was t,he fact’ that under the wnditions used, t,hcre was no rc~lcasc of DNA or detwtable amounts of DKA4-dqwndent ILTAI polynwrase. Sinw ATI’ has bwn rcyortttd (21, 22) to stimulate t,lw rclcnse of’ viral RX*% from infrcted IiB ~(41 nuclei, ~vc adwovirus-2 this s\km. l’rehW also inwstigntod liminar>- results showed the same pat8tcrn of RX:! relcasc from the adonovirus-2infected HcT,a nuclei as ohscrved in t,he uninfcct,cd nuclei, namely that I\Ig’- inhibits RSAI rrlcaw from \-irus-infected nuclei, and this inhibition van be rcvcrsed by ATP. ‘I& prcwnt8 results indicat,t: that the Ilrlc,lcot,id~,-dcE,ctndcnt release of RSA from l~uclvi that, has been reportcld in sweral s>-,stcms (15-Z), ma!: bc inwAy due to the :tl)ility of the nwleotldt: to rhclatc l\I$+ as first suggcstcd by Schrwider (15).
K., S.YM.LRIK.~, (19i2) Satztrc 1%. 9.
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J. 3101. Biol.
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[‘A.1 58, 10% -1094.