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Cell, Vol. 27, 487-496, December 1981 (Part 2), Copyright 0 1981 by MIT

In Vitro Splicing of the Ribosomal RNA Precursor ofTetrahymena: Involvement of a Guanosine Nucleotidein the Excision of the Intervening SequenceThomas R. Cech, Ar thur J . Zaugand Paula J. GrabowskiDepartment of Chem istryUniversi ty of ColoradoBoulder, Colorado 80309

SummaryIn previous studies of transcr ipt ion and spl ic ing ofthe r ibosomal RNA precursor in isolated Tetrahy-mena nuclei , we found that the intervening se-quence ( IVS) was excised as a unique l inear RNAmolecule and was subsequently cycl ized. In thepresent wo rk, transcr ipt ion at low monovalent cat-ion conce ntration is found to inhibit splicing and tolead to the accumulation of a spl ic ing intermediate.This intermediate contains spl ic ing activi ty thatei ther is t ightly bound to the RNA or is part o f theRNA molecule i tsel f. The intermediate is able tocomplete the excision of the IVS when i t is incu-bated w ith a monovalent cation (75 mM (NH&S04),a divalent cation (5-10 mM MgC12) and a guanosinecompound (1 gM GTP, GDP , GMP or guanosine).ATP, UTP, CTP and guanosine compound s without2 and 3 hydro xyl groups are inactive in causingexcision of the IVS. Accurate excision of the IVS,cycl ization of the IVS and (apparently) l igation ofthe 26s rRNA sequences bordering the IVS al l takeplace under these condit ions, suggesting that asingle a ctivity is responsible for all three re actions .Dur ing excision of the IVS, the 3 hydroxyl of theguanosine moiety becomes l inked to the 5 end ofthe IVS RNA via a normal phosphodiester bond.When GTP is used to dr ive the reaction, i t is addedintact without hydrolysis. Based on these resul ts,we propose that Tetrahymena pre-rRNA spl ic ingoccurs by a phosphoester transferase mech anism.According to this model, the guanosine cofactorprovides the free 3 hydroxyl necessary to ini t iate aser ies of three transfers that resul ts in spl icing ofthe pre-rRNA and cycl ization of the excised IVS.IntroductionMany eucaryotic tRNA , rRNA and mRN A genes areinterrupted by stretches of noncoding DNA cal ledintervening sequences ( IVSs), which are transcr ibedas part of a precursor RNA and are subsequentlyremoved by a cleavage- ligation reaction termed spl ic-ing. Al though no spl ic ing enzym e has yet been pur i-f ied, some m ajor features of the reaction mechan ismhave been descr ibed for the spl ic ing of yeast tRNAprecursors. In that case, splic ing is accompl ished byendonucleolyt ic excision of the IVS and ATP-depen-dent l igation of the two resul t ing tRNA hal f-molecules(Knapp et al ., 1979; Peebles et al ., 1979). The cleav-age and l igation activi t ies have been separated and

partially purified, and appear to reside in differentenzym e molecules (C. Peebles and J. Abelson, per-sonal comm unication). I t has heretofore been un-known whether the spl ic ing of mR NA and rRNA pre-cursors might fol low pathways simi lar to that used fortRNA precursors, al though from comparison of thenucleotide sequences at spl ice junctions i t appearsthat at least the compone nts that recognize the IVSare di fferent for the three c lasses of transcr ipts.In Tetrahymena pigmentosa strain 6 UM and in T.thermophi la, al l copies of the rDNA (rRNA gene) con-tain a 0.4 kb IVS (Cech and Rio, 1979; Din et al .,1979; Wi ld and Gal l , 1979). When isolated Tetrahy-mena nuclei are incubated under condit ions that al lowelongation of rRNA chains ini tiated in vivo, the rRNAprecursor is synthesized and spl iced (Zaug and Cech,1980). T ranscription and splicing also occu r in iso-lated nu cleoli (Carin et al., 1980). The IVS is excise dfrom the pre-rRNA as a l inear 0.4 kb RNA moleculeand is subsequently converted to a circular RNA (Gra-bowski et al . , 1981). The same splic ing pathway ap-pears to occu r in vivo, w ith both linear and circularforms o f the excised IVS being present in nuclear RNA(S. Brehm and T. Cech, manuscr ipt in preparation).

We have recently analyzed the sequence of theexcised IVS RNA and have compared i t wi th thesequence of the corresponding region of the rDNA,determined by N. Kan and J. Gal l (manuscr ipt inpreparation). Both RNAase Tl f ingerpr int analysis ofuni formly labeled IVS RNA and sequence gel analysisof Y-end-labeled IVS RNA led to the same conclusion:the IVS RNA has a 5 terminal pG (guanosine 5-monophosphate) not found in the DNA sequence, andthereafter has a sequence col inear with that of therDNA (A. Zaug and T. Cech, manuscr ipt in prepara-tion).

We now present evidence that excision of the IVSrequires a guanosine residue, which is added to the5 end of the IVS dur ing excision. We propose thatthis is the f i rst in a ser ies of phosphoester transferevents that ul t imately leads to the spl ic ing of theTetrahymena rRNA precursor and the cycl ization ofthe excised IVS. Such a mechan ism is completelydi fferent from that involved in the spl ic ing of yeasttRNA precursors.ResultsTrapping a Splicing Interm ediateThe sal t dependence of transcr ipt ion and spl icing ofpre-rRNA in isolated nuclei is show n in Figure 1.Transcr ipt ion proceeds at low concentrations of mon-ovalent cation, but release of the IVS RNA is almosttotal ly inhibi ted. When the RNA synthesized in low sal twas subjected to heat denaturation or treated withproteinase K, no addi t ional IVS RNA was released(Figure 1). In other exper iments, more extensive treat-ment of the RNA with proteinase K or pronase, or

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Cel l488

(A(NI mMFigure 1. Salt Dependence of Transcriptionand Splic ing of Pre-r ?NA in Isolated T. ther-mophi la Nuc le i(A) The rRNA transcribed at different(NH&SO, concentrations was analyzed by2.5%-i 5% polyacrylamide gradient gel elec-trophoresis. Samples containing eq ualamounts of 32P radioactiv ity were either lay-ered directly onto the nonden aturing gel (Na-

IVS F NA-

(B) -41 ,100I I75 +Ii 50 2

1s-\ \ 25 5\ . Ii

2300 400 0

(NH4)2 SO4 , mM

electrophoresis of the RNA on an 8 M urea, polyacryl-amide gel at 65C, fai led to increase the amount offree IVS RNA (data not shown). I t wi l l become clearfrom the resul ts presented below that the IVS is st i l lpresent in the high molecular weight RNA synthesizedunder these condit ions. We do not know whether theIVS is stil l an integral part of the precursor, or isassociated with it by means o f another molecule suchas a spl ic ing enzym e.

We hoped to use the unspl iced pre-rRNA synthe-sized in vi tro under low sal t conditions as a substrateto assay for spl ic ing activi ty in nuclear extracts. Thisapproach proved to be impossible, however, becauseexcision of the IVS occurred when the isolated RNAwas incubated in transcr ipt ion cocktai l (see Exper i-mental Procedures) at normal sal t concentration in theabsence of a nuclear extract. The pre-rRNA isolationprocedure included two SDS-phenol extractions, sed-imentation in a formamide-sucrose gradient, and insome cases protease treatment, so the presence ofsuch activi ty in the RNA preparation was completelyunexpected. The smal l RNA molecule produced in thisautoexcision reaction was identif ied as the IVS RNAby i ts electrophoretic mobi l i ty relative to an IVS RNA

marker, by i ts conversion to the circular form when

-23s-16s

t ive) or were f irst boiled for 5 min in electro-phoresis buffer and cooled on ice (Heat-de-natured). (Lane 57 RNA transcribed in 5 mM(NH&SO, was subsequently treated with pro-teinase K to destroy any protein-RNA com-plexes that might have prevented release ofthe IVS. (Lane E. coli) rRNA size markerslabele d with polynucleotide k inase.(6) Quantitation of transcription and IVS exci-s ion. 10% of the total radioactiv ity released asfree IVS RNA is taken to correspond to 100%excis ion (Zaug an d Cech, 1980).

the reaction was performed at 39C (Figure 2) and bydirect sequence analysis (see below).Inhibition of splicing in isolated nuclei thereforeleads to the accumulation of a spl ic ing intermediate

that can complete the excision of the IVS under favor-able condit ions. The activi ty of this intermediate maybe due to a spl icing enzym e tightly bound to the pre-rRNA, or i t may be a novel case of an RNA-mediatedreaction that requires no protein (see Discussion). Inaddit ion to IV&excision activi ty, the intermediate hasat least one type of l igation activi ty: that responsiblefor cycl ization of the excised IVS RNA . The pre-rRNAalso appears to be l igated in this system, as judgedby the absence of a discrete 1070 nucleotide RNAfragment (extending from the 3 end of the IVS to the3 end of the pre-rRNA) that would be produced byexcision of the IVS without l igation of the resul t ingpre-rRNA hal f-molecules. I f such a fragment wereformed, i t would be clear ly resolved by gel electro-phoresis (see, for example, Figure 2).Requirements for IVS ExcisionThree compone nts of the transcr ipt ion cocktai l (seeExper imental Procedures) proved to be necessary tocause excision of the IVS from the spl ic ing interme-diate. These compone nts were a monovalent cation,a divalent cation and GTP. We determined the opti -mum concentration of each cofactor empir ical ly byincubating the splicing intermed iate (purified as de-scr ibed in Exper imental Procedures) wi th the threecofactors, two of which were held at f ixed concentra-t ion whi le the concentration of the third was var ied.The products of the IVS-excision reaction in eachcase were analyzed by denatur ing gel electrophore-sis. One example, the magnesium ion dependence, isshown in Figure 3A . The percentage of IVS excisedwas calculated from data obtained from densi tometr ictracings of the autoradiographs (Figures 3B, 3C and

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Guanosine Involved in RNA Splic ing489

h MgC I ,&@I / I / 5mM IOmMnt bRI 3039M -0ri21934-

1373-947- *832.565- **r

we4 -Cv,_ _- c -L

Figure 2. Transcription under Low Salt Condit ions Produces an Un-spliced Pre-rRNA That Is Active in Splic ing and IVS Cyclization inVitroThe splic ing intermediate , labele d with 32P during transcription, waspurif ied as described in Experimental Procedures. It was then incu-bated with GTP and (NH&SO4 at two concentrations of MgC l* andtwo temperatures, as indicated. The RNA products were analyzed bydenaturing gel electrophoresis at 65C. Denatured lambd a DNArestr ic tion fragments produced by a Hind Il l-Eco RI doub le digestwere used as molecular weight markers ( left lane). (Lane M) Markersfor the l inear(L) and c ircular(C) IVS were purif ied from RNA producedby high salt transcription in isolated nuclei as previously described(Grabowski et al., 1981).

30). Excision of the IVS occurred at a maximal levelw i th 575 m M (NH&SO+ 5-10 mM MgCl* and 21 pMGTP.

The circular IVS is also formed under these condi-t ions. Cycl ization is enhanced at 39C compared to30C as shown in Figure 2, consistent wi th our ear lierobservations that IVS cycl ization is temperature-de-pendent in vi tro (Grabowski et al . , 1981). The cycl i-zation reaction has an absolute requirement for mag-nesium ion (Figure 3B; our unpubl ished observations),and is inhibited by high concentrations of ammo niumsulfate (Figure 3D).We found that ATP, CTP and UTP at concentrationsas high a s 10 ,uM did not substi tute for GTP in thereaction (data not shown). (Some reaction was ob-served with 1 mM ATP, C TP or UTP , which could beexplained by a 0.01 %-0.10% contamination of GTPin these other nucleotides.) Several guanosine com-pounds other than Y-GTP were active as cofactors inexcising the IVS in vitro (Figure 4; Table 1). Guano-sine, 5-GMP and 5-GDP showed simi lar levels ofactivi ty in the reaction compared with 5-GTP (Table2). Compoun ds lacking a 2 or 3 hydroxyl , as in thecase of 2-deoxyGTP, 2 ,3-dideoxyGTP and 3-GMP,were inactive as cofactors in the reaction.

We therefore conclude: a guanosine compoundcontaining both a free 2 and a free 3 hydro xyl isrequired for excision of the IVS in vitro; and theguanosine compound required for the reaction is notuti l ized as an energy cofactor. That is, since thenumber of phosphates at the 5 posi t ion of the gua-

nosine moiety can be var ied from 0 to 3 without anyobservable change in the amount of IVS excised,phosphate or pyrophosphate cleavage is unl ikely tobe important in the reaction.

The t ime course of IVS excision was determined,for which the autoradiograph of the denaturing gel isshown in Figure 5A. The data were quanti tated bothby densi tometr ic tracing o f the autoradiograph and byscintil lation counting of the sliced, dried gel (Figure5B). The data obtained by the two different methodsagreed favorably. As shown in Figure 5B, the reactionwas hal f complete after 2 min. The maxim um amountof IVS excision occurred after 10 min under theseexper imental condit ions, which justif ies the use of 30min incubation t imes for the exper iments descr ibedabove.Addit ion of a Guanosine Nucleotide to the 5 Endof the IVS dur ing i ts ExcisionWe tested the possibil i ty that the guanosine requiredfor IVS excision is the source of the extra G residuefound on the 5 end of the excised IVS RNA . W eprepared the pre-rRNA splicing interme diate by invitro transcription using H-UT P a s the labeled pre-cursor. Gel electropho resis followed by tritium fluo-rography (Figure 6A) revealed no free IVS RNA in thepreparation.

Whe n the splicing interme diate (either 3H-labeled orunlabeled) was incubated with (Y-~P-GTP under thecondit ions for IVS excision descr ibed above, 32P ra-dioactivi ty was bound only to one RNA species, whichhad the electrophoretic mobi l i ty of the excised l inearIVS (Figure 6B). Southern hybr idization of this RNA toBgl II , Hae I l l , Hha I and Hind I l l restr ict ion fragmentsof rDNA confi rmed that i t was the IVS (data n otshow n). No labeling of the IVS occurred in parallelincubations, in which a-32P-ATP w as substi tuted forthe GTP or in which Mg2+ was excluded from thereaction. Each sample contained a large amount ofunincorporated nucleoside tr iphosphate, a smal l frac-t ion of which remained at the or igin. In no case wasthere any detectable labeling of the pre-rRNA, whichmigrated wel l into the gel (Figures 6B and 6C). Whenthe spl ic ing intermediate was incubated with 32P-GMP,the IVS RNA was again excised and labeled (data n otshown).The IVS RNA labeled dur ing completion of spl ic ingin vi tro was recovered from polyacrylamide gels andanalyzed by digestion with var ious enzy mes (Figure7). Treatment of the GMP-labeled IVS RNA with bac-terial alkaline phosp hatase led to the release of thelabel as inorganic phosp hate, indicating the terminallocation of the label . Treatm ent of the RNA with nu-clease Pl produced labeled pG, whi le treatment wi thRNAase Tl or T2 produced labeled pGp. Dur ing spl ic-ing, therefore, the labeled GMP becam e l inked to the5 end of the IVS RNA via a normal phosphodiesterbond (3 hydroxyl of GMP to 5 phosphate of IVS

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Cel l490

A) B)6.01MgCI2, mM

M 1 2 5 10 15 25 50~-o,,

C)

601

GTP, M0 40 80 120 160

(NH4&,S04, mMFigure 3. Three Cofactors Are Required for Excis ion of the IVS from the Purif ied Splic ing Intermediate(A) Denaturing gel electrophoresis of the RNA products of the IV&-excis ion reaction at increasing concentrations of MgCI?, with the GTP and(NH&SO4 concentrations held constant.(B) Data obtained from densitometric tracings of the autoradiograph in (A) give the MgC12 dependen ce of IVS excis ion in v itro. The GTPdepende nce (C) and the (NH&SO4 dependen ce (D) of the reaction were determined in analogous experiments. (U) Total excised IVS (l inearand c ircular). (A--A) Excised linear IVS. (W-1) Excised c ircular IVS. Data are given as a percentage of the total RNA in each sample.

RNA ). The same type of analysis was performed onthe IVS RNA labeled with ol-32P-GTP dur ing completionof spl ic ing. In this case, treatment with nuclease Piled to the release of the label as pppG (data notshown). The GTP, l ike the GMP , was therefore l inkedto the 5 end of the IVS RN A via a normal phospho-diester bond. The data el iminated the possibi l ity thatthe guanosine nucleotide is forming a cap structuresuch as that found on smal l nuclear RNA s, as wel l asthe possibi l ity that hydrolysis of GTP accompan iesformation of the covalent bond.The fraction of the excised IVS RNA molecules thatwere end-labeled with GTP during splicing wa s cal-culated from the 32P to 3H ratio of the IVS RNA. Forexample, gel sl ice 13 in Figure 6C contained 1 1,956cpm 32P (7.2 x 1 O- l5 mole GTP) and 14,000 cpm 3H(7.3 X lOTi5 mole IVS RNA , based on 107 ur idinesper molecule [N. Kan and J. Gal l , manuscr ipt in prep-aration]). The molar ratio was therefore 7.2/7.3 =0.99 GTP/IVS RNA . In another spl ic ing reaction, theratio was 1.35. We conclude that the excision of each

IVS RNA molecule is accompanied by the addi t ion ofa guanosine nucleotide to i ts 5 end.When the spl icing intermediate was incubated un-

der higher temperature condit ions that promote bothexcision and cycl ization of the IVS, a smal l amou nt oflabeled l inear IVS RNA was observed, but no labeledcircular IVS RNA was observed (Figure 6B lane HI. Inother exper iments in which ei ther GTP or GMP wasused, labeling of the l inear IVS RNA was obtained at39C, but in no case was label detected in the gel atthe posi t ion of ci rcular IVS RNA . These resul ts provideprel iminary evidence that the guanosine nucleotideadded to the 5 end of the l inear IVS RNA dur ingexcision is spl i t off dur ing cycl ization.Accuracy of IVS Excision in Vi troWe prepared 5-end- labeled IVS RNA by al lowingisolated spl ic ing intermediate to complete excision ofthe IVS in vi tro in the presence of 3P-GTP. Its nucleo-t ide sequence was then determined by part ial diges-t ion with base-speci f ic RNAa ses fol lowed by gel elec-

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y;snosine Involved in RNA Splic ing

Figure 4. The IVS-Excis ion Reaction Requires a Guanosine Cofactorwith a Specif ic StructureDenaturing gel electrophoresis of the RNA products of the IVS-exci-s ion reaction, in which various guanosrne compounds were tested fortheir activ ity as cofactors in the reaction. All compounds shown weretested at a f inal concentration of 2 PM. ddGTP: 2,3-dideoxyGTP. G:guanosine. dG: P-deoxyguanosine. (Lane HzO) A control in whichwater was substituted by volume for the guanosine cofactor.

trophoresis (Donis-Kel ler et al . , 1977; Simoncsi ts etal ., 1977). From the sequencing gel (Figure 8A) 26 ofthe f i rst 31 nucleotides could be identi fied. The RNAsequence was in total agreement with the DNA se-quence of the IVS, except for the 5 terminal G addedto the RNA dur ing spl ic ing (Figure 8B). Thus the IVSthat wa s end-labeled during excision in vitro has adiscrete 5 end that is exactly the one expected to beproduced by accurate spl ic ing. Furthermore, this ex-per iment confi rms that the GTP is l inked directly tothe RNA : the labeled products of the var ious RNAasetreatments have the correct electrophoretic mobi l i t iesfor ol igonucleotides, which would not be the case i fthe labeled GTP were l inked to a protein associatedwi th the RNA.DiscussionWe have used transcr ipt ion in isolated nuclei underlow sal t condit ions to accumulate unspl iced pre-rRNA.This RNA , puri f ied by methods that normal ly resul t incomplete deproteinization, has the inherent abi l ity toexcise i ts own IVS. A monovalent cation, a divalentcation and a guanosine compound are necessary andsuff ic ient to cause the excision. Wha t is the nature ofthe activi ty responsible for excision of the IVS andconcom itant attachme nt of a guanosine nucleotide toi ts 5 end? Either these events are catalyzed by aprotein bound to the RNA , an unusual protein thatretains activi ty through SDS-phenol extraction, boi lingand protease treatment; or the events are mediatedby the RNA molecule i tsel f. We examine these possi-bil ities in more detail in the next sectio n.The guanosine compound required for IVS excisionbecome s covalently attached to the 5 end of the

Table 1. Activity of Various Compoun ds in the Release of the IVSfrom the Pre-rRNA Splic ing IntermediateActive Inactive5-GTP 5-ATP. 5-CTP. 5-UTPS-GDP Guanine5-GMP 3-GMPGuanosine 2-Deoxyguanosine, P-deoxyGTP

2,3-DideoxyGTPActiv ity was measured at a f inal concentration of 2 @M.

excised IVS RNA . It is not joined tot@- RNA by aphosphoanhydr ide bond, such as that formed by AMPin the adenylated nucleic acid intermediates producedby DNA l igase or RNA l igase (Kaufmann and Li ttauer,1974; Lehman, 1974). Instead, based on the suscep-t ibi li ty of the l inkage to cleavage by RNAa ses Tl andT2, the 3 end of the guanosine moiety mu st form anormal phosphodiester bond with the 5- terminaladenosine of the IVS. Presumably the phosphategroup of the bond is contr ibuted by the adenosine.When GTP is used as the cofactor, the excised IVSRNA has the sequence Y-pppGpA . . ; when GMPis used, the IVS has the sequence 5-pGpA . . TheIVS produced by linked transcription-splicing in iso-lated nuclei has a 5- terminal monophosphate (A.Zaug and T. Cech, manuscr ipt in preparation). Thepresence of the monophosphate could reflect a pref-erence for GMP as the cofactor for spl ic ing in nuclei .Al ternatively, spl ic ing could involve addi t ion of GTP tothe IVS RNA , with cleavage of inorganic pyrophos-phate occurring later.

The circular form of the IVS RNA was or iginallyfound as a product of transcr ipt ion and spl ic ing inisolated nuclei , and i t was not known whether cycl i -zation was part of the spl ic ing process or the resul t ofan unrelated RNA-l igase activi ty in the nucleus (Gra-bowski et al . , 1981). We now find that when pur i f ied,uniform ly labeled pre-rRNA splicing intermed iate isallowed to continue splicing in vitro, the circular aswel l as the l inear form of the excised IVS RNA isproduced. Cycl ization therefore appears to be per-formed by an activi ty involved with spl ic ing, and mayin fact be an obl igatory step in the process (see themodel below).RNA spl ic ing requires both excision of the IVS andl igation of the resul t ing exons (mature RNA sequencesthat were interrupted by the IVS in the precursor) .Incubation of the pre-rRNA spl ic ing intermediate withthe necessary cofactors resul ts in excision of the IVS.Evidence for exon l igation, however, is indirect, basedon the lack of a 3 exon fragment that would beproduced by cleavage in the absence of l igation. Inthe low sal t condit ions we used for synthesis of thepre-rRNA, accurate termination of transcr ipt ion oc-curs (Leer et al ., 1979); the 3 exon should therefore

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Table 2. Comparison of Guanosine CofactorsIVS Excised (%)2.0 pM 0.2 PM Increase in IVS

Cofactor (a) (b) Excision a/bGTP 4.9 2.7 1.8GDP 4.5 2.7 1.6GMP 4.3 2.7 1.6Guanosine 8.6 5.6 1.5Mean f SD 1.6 f 0.1Each guanosine compound was tested at two final concentrations,the concentration at which GTP gives maxim al IVS excis ion (a) andthe,Loncentration at which GTP gives half-maxima l excis ion (b).Vafues have been corrected for the 1% IVS in the control, in which an

.I &al volume,of water was substituted for the cofactor.

be a discrete size fragment. Based on recent mappingof the 3 end of the pre-rRNA at the nucleotide se-quence level (N. Din, J. Engberg and J. Gal l , personalcomm unication), the size of this RNA would be 1070nucleotides. Because gel electrophoresis (see, forexample, Figure 2) revealed no such RNA , i t is l ikelythat the spl ic ing intermediate is also capable of exonl igation. Al ternatively, the two exons may be heldtogether in the spl ic ing complex without having beenl igated. A more direct test of l igation may be providedby Sl -nuclease-protection studies of hybr ids formedbetween the in vi tro spl ic ing product and the rDNA.Structure of the Spl icing IntermediateThe speci f ic i ty of the reactions performed by thespl ic ing intermediate, and the absolute dependenceof these reactions on a guanosine compound , makeit seem very likely that an enzyme is associated withthe RNA . The activi ty is not destroyed or removed,however, when the spl ic ing intermediate is subjectedto the fol lowing treatments: boi ling in the presence ofSDS and 2-mercaptoethanol, SDS-phenol extractionat room temperature or at 65C sedimentation in a60% formamide sucrose gradient or treatment withproteases according to a var iety of protocols de-scr ibed in Exper imental Procedures. Al though suchresul ts are normal ly taken as strong evidence againstprotein involvem ent, some known nucleic acid en-zyme s are resistant to inactivation by heat, SDS orprotease treatment. For example, the single-strand-speci f ic en donuclease activi ty of Bal 31 nuclease ishighly resistant to digestion by pronase (1 mg/m l for10 min at 37C; H. B. Gray, Jr . and C. F. Wei, personalcomm unication) and persists in the presence of 5%SDS when incubated in a high sal t buffer containingcalcium and magnesium (Gray et al ., 1975). Further-more, attachme nt of a protein to a large RNA m oleculecould a ccount for i ts surviving the phenol extraction,and might explain i ts resistance to protease. Attemp tsto detect a protein on the spl ic ing intermediate bylabeling with radioactive amino acid s are in progres s.

A) T IME C OU R SE0 15' 36' 2' 5 10' 30 M D S -Or,

B)30-

2.5-QI$ 2.0-G5 I5

05L----5 10 15 20 25 30TIME (min)Figure 5. Time Course of the IVS-Excis ion Reaction(A) Denaturing gel electrophoresis of the RNA products of the IVS-excis ion reaction monitored as a function of t ime. (Lane S) Theuntreated starting m aterial. (Lane D) 32P-end -labeled DNA marker.(B) The amount of excised IVS RNA, given as a percentage of thetotal labele d RNA, was determined from densitometric tracings of theautoradiograph in (A) (M). and by scinti l lation counting o f thesliced, dried gel (u.

The resistance of the spl ic ing activi ty to phenolextraction, SDS and proteases can also be interpretedin a more straightforward manner. The rRNA precur-sor might be able to undergo spl ic ing without thepart ic ipation of a protein enzym e. A port ion of theRNA chain could be folded in such a way that i t formedan active si te or si tes that bound the guanosine cofac-tor and catalyzed the var ious bond-cleavage and I i -gation events. I f one of the RNA molecules producedin the reaction ( for example, the free IVS) retained i tsactivi ty and catalyzed addit ional spl icing even ts, theni t would be an example of an RNA en zyme.

Another unresolved question about the splicing in-termediate concerns the means b y which the IVS isattached to the rest of the pre-rRNA. The IVS was notreleased from the high molecular weight RNA by anyof the treatments mentioned in the preceding para-graph, or by electrophoresis in an 8 M urea gel at65C. These resul ts provide strong evidence that the

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Guanosine Involved in RNA Splic ing493

(A) (B)

- Ori -

Gel slice numberFigure 6 . Addit ion of a Guanosine Nucleotide to the IVS during Its Excis ion(A) Pre-rRNA splic ing intermedia te synthesized in nuclei under the low salt condit ions with H-UTP as the labele d precursor. RNA was subjectedto electrophoresis on a 4% polyacrylamide, 8 M urea gel, which was then subjected to tr it ium fluorography. Note the absence of free IVS RNA.(6) Splic ing intermediate ( isolated as in [A], but unlabe led) was incubated with 32P-GTP or P-ATP. (Lane a-32P-GTP) RNA incubated with 40 &iw~*P-GTP (5 FM) for 30 min at 3OC in 10 mM MgCl *, 100 mM (NH&SO, and 40 mM Tris (pH 7.5). RNA splic ing intermedia te was at aconcentration of -1 nM. (Lane w~*P-ATP) Same condit ions, with GTP replaced by 40 $i ATP. (Lane H) a-32P-GTP incubation at 39OC instead of30C condit ions that favor cyclization of the excised IVS. (Lane EDTA) same as a-32P-GTP incubation, except MgC12 replaced by EDTA. The twolanes to the r ight show the reaction with a different preparation of pre-rRNA splic ing intermediate. (Lane M ) Markers for the pre-rRNA and thecircular and linear IVS, which were produced from the splic ing intermediate (uniformly labele d with 32P during transcription) by incubation asdescribed above, except with 1 mM unlabe led GTP.(C) Trit iated splic ing intermediate shown in (A) was allowed to complete excis ion of the IVS in the presence of ~I-~>P-GTP. After gel electrophoresis,the eel was dried and s liced in to one-eighth-inch fractions. The s lices were solubil ized. and the H (0 - - 0) and 32P (x-x) radioactiv it ies weredetermined by l iquid scinti l lation counting

25-20- f\,

,15- IT-JO- I \ m-0T-

Pu-PG-

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-PGP-PAP-PCP

-0ri.12345678

Figure 7. End Analys is of IVS RNA Labeled w i th 5 -GMP dur ingExcis ionTrit iated splic ing intermed iate was incubated with 32P-GMP. salt andMgC12 to allow excis ion of the IVS to be completed. The IVS producedduring this reaction was purif ied by polyacrylamide gel electropho-resis. It was then analyzed by high-voltage paper electrophoresisafter no enzymatic incubation ( lane 1), or after digestion to completionwith nuclease Pl ( lane 3). bacterial alkaline phosphatase ( lane 4) orRNAase T2 ( lane 5). (Lane 2) Uniformly labele d IVS RNA digestedwith nuclease Pl provided markers for the four pNs. (Lanes 6, 7, 8)Markers for the four pNps. as indicated.

IVS is covalently bound to the pre-r-RNA. The simplestpossib il ity is that the IVS is stil l an integral part of thepre-rRNA polynucleotide chain. Because there maybe an unusual ly stable protein associated with thesplicing intermed iate (see above ), it is also possiblethat the IVS has already been part ial ly excised fromthe pre-rRNA and remains bound to i t v ia a proteinl inkage ( for example, as shown in Figure 9 A). Suchan intermediate would be simi lar to the covalent com-plex of a DNA topoisomerase with DNA that is formedwhen a nicking-closing reaction is interrupted in vitro(Depew et al ., 1978).A Model for Pre-rRNA Spl icingThe seemingly incongruous features o f pre-rRNAspl icing include the addi t ion of guanosine to the IVSdur ing excision, the cycl ization of the IVS, the appar-ent lack of an ATP requirement for cycl ization or exonl igation and the possibi l ity that a single activi ty accom-pl ishes excision of the IVS and the two l igations.These features are largely reconci led by the modelpresented in Figure 9. According to this model, thespl ic ing enzyme is a phosphoester transferase. Theguanosine cofactor provides a free 3 hydroxyl , towhich the 5 end of the IVS is transferred. A secondphosphoester transfer releases the IVS and joins thetwo exons, whi le a third transfer cycl izes the IVS andreleases the enzyme and the guanosine cofactor. This

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(A)

G

(B)ONA: ~:.CTCTC~~SIATAGCAATR~TTACCTTTGGAGGGAAAA~...CTCG~AAGGT.~:IVS RNA: PPPGAAANAGNAANAUUUNCCUNUGGAGGGAAAA...Figure 8. Nucleotide Sequen ce Analysis of the IVS RNA Labele d withGTP during Excis ion(A) IVS RNA was prepared as described in the legend to Figure 7,except that the labele d cofactor was ly-32P-GTP. T he IVS RNA wasthen digested to various extents with RNAase Tl (cleaves after G),U2 (c leaves after A ) or Phy M (c leaves after A and U), and subjectedto electrophoresis on a sequencing gel. (Lanes OH-) Partial alkalinehydrolysis produced a ladder showing the posit ions of all theoligonucleotide s. (Lane -ENZYME) RNA incubated without RNAasewas cleaved to some extent, preventing the identif ication of some ofthe nucleotides (N). The diffuse band formed by the mononu cleotide(pppGp) is most easily seen in the RNAase Tl digest that proceededalmost to completion.(B) The RNA sequence determined from the gel in (A) is comparedwith the sequence of the IVS region of the rDNA (N. Kan and J. Gall,manuscript in preparation). Arrows: the IVS boundaries in the DNA.

model invokes a single en zymatic activ i ty to accom-pl ish excision and cycl ization of the IVS and l igationof the exons, thereby simpl i fy ing the problem of howal l three reactions could be performed by the activi tyassociated with the spl ic ing intermediate. Because

(B)

4(0 PG- t ---L.-iIMg+2 I

Figure 9. A Strand-Transfer Mod el for RNA Splic ing(A) The diagrams show two of several possible structures for theintermediate that accumulates when the splic ing reaction is inhibite dby low salt concentration. Solid l ines: pre-rRNA exons. Wavy line: theIVS. Dashed rectangle: the splic ing enzyme (a protein or a doma in o fthe RNA). In the structure to the left, the enzyme is t ightly bound tothe substrate RNA; in the structure to the r ight, the enzyme has madeone chain scission and is covalently bound to one or both of thetermini. All s trand c leavages produce 3 hydroxyl (0) and 5 phos-phoryl(0) termini.(B) In the presence of the guanosine compound (shown as 5-GMP)and monovalen t and divalent cations, the f irst phosphoester-transferreaction is completed. The 5 end of the IVS RNA is l inked to the 3hydroxyl of the guanosine cofactor.(C) A second phosphoester transfer involves c leavage of the RNA atthe 5 end of the distal exon and rejoining of this phosphate to the 3hydroxyl of the proximal exon. S plic ing of the pre-rRNA is nowcomplete. The IVS is released as a l inear mo lecule with an added 5-terminal g uanosine nucleotide (see text). Cyclization activ ity is t ightlyassociated with the excised IVS (P. Grabowski, unpublish ed results).(D) A third phosphoester transfer involves c leavage of the IVS near its5 end and rejoining with a new 3 hydroxyl group. In this case, th e 3hydroxyl group is at the end of the same strand of RNA, so that thereaction produces a c irc le. Recent results indicate that the guanosinenucleotide added during excis ion is released upon cyclization; it isnot released as a free monon ucleotide, but as part of a larger moleculethat has not yet been identif ied (A. Zaug. unpublishe d results).Release of splic ing activ ity could occur only if i t were a separatemolecule.cleavage and l igation are l inked, the proposed mech-anism circum vents the need for ATP or GTP hydro-lysis.

The l inear excised IVS RNA has a 3- terminal G-OH(A. Zaug and T. Cech, manuscr ipt in preparation).Cycl ization of the IVS, l ike the f i rst phosphoestertransfer event, therefore involves an attack by the 3hydroxyl of a guanosine residue. A guanosine at the3 end of the IVS is a feature comm on to rRNA andmRN A intervening sequences (see, for example, Ler-ner et al ., 1980; Wi ld and Somm er, 1980; Nom iyamaet al ., 1981). I t is possible that this guanosine isconserved because i t is required for IVS cycl ization,though the only other case in which cycl ization hasyet been observed involves yeast mitochondr ial mRN Aintervening sequences (Arnberg et al ., 1980; Hal-breich et al ., 1980).

The proposed phosphoester- transferase activi ty issimi lar to that of DNA nicking-closing enzym es, ex-cept that the 5 phosphate produced by the nickingstep is joined to a di fferent 3 hydroxyl group. DNA

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nicking-closing events catalyzed by E. col i DNA to-poisomerase I or the NalA subunit of DNA gyrase haveno energy requirement (Kornberg, 1980). The phos-phoester transferase also bears some simi lar i ty to theA and A* proteins involved in @Xl 74 DNA repl ication(Eisenberg et al ., 1977; van der Ende et al ., 1981). Inboth cases, the enzyme introduces a nick at a speci f icplace in a nucleic acid molecule, producing a 3hydroxyl ; remains bound to the nucleic acid; and whi lebound can catalyze a cleavage- ligation reaction thatresults in production of a circular nucleic acid mole-cule.

The model does not address the problems of rec-ognit ion of the IVS and formation of the spl ic ing com-plex. These processes may be mediated by an adapterRNA such as a tRNA (Gourse and Gerbi , 1980) or asmal l nucleolar RNA (Reddy et al ., 1979; Wise andWeiner, 1980) both of which have some sequencecomplem entar i ty to exon sequences on both sides ofthe P/S.Experimental ProceduresNucleotides and EnzymesUnlabe led nucleoside tr iphosphates were purchased from P-L Bio-chemicals, and for some experiments were further purif ied as de-scribed by McClure et al. (1978). Other nucleotides, nucleosides andbases were of the highest quality available from Sigma an d were usedwithout further purif ication. P-labeled nucleoside hiphosphateswere purchased from New Engla nd Nuclear. Labele d nucleoside3,5-bisphosphates (pNps) were made from the corresponding un-labele d 3-monophosphates with polynucleotide k inase and r3*P-ATP (both from New Engla nd Nuclear). We made 5-32P-GMP frompGp using nuclease Pl (Sigma) to remove the 3 phosphate; it wasthen purif ied bythin-layerchromatographyon PEI-cellulose. RNAasesTi. T2 and U2 were obtained from Sankyo, and bacterial alkalinephosphatase was obtained from Bethesda Research Laboratories.RNAase Phy M was a gift from N. Pace.Transcription and Splic ing in Isolated NucleiGrowth of cells (T. thermoph ila strain B VII) and isolation of nucleiwere carried out as described by Zaug a nd Cech (1980). Transcrip-t ion reactions contained 1 to 2 X 10 nuclei in 0.45 ml of transcriptioncocktail (53 mM Tris-HCI [PH 8.01, 5.3 mM MgC& , 1 mM Car&, 1mM putrescine, 1 mM spermidine, 0.1 mM spermine, 2 mM 2-mer-captoethanol, 0.048 mM each of ATP, CTP, UTP and GTP, 0.2-0.4mM aurintr icarboxylic acid (nuclease inhibitor) and 40 pa/ml ol-am an-it in). In most experiments one or all four of the nucleoside tr iphos-phates were either a-32P- or $H-labeled . In addit ion , each reactioncontained (NH&SO4 at a concentration of 5 mM to prevent splic ingor 120 mM to optimize the excis ion of the IVS. The mixture wasincubated for 30 min at 30C with occasional shaking, at which timethe reaction was stopped by the addit ion of EDTA and SDS to f inalconcentrations of 20 mM and I%, respectively. The incorporationvaried from 2%-20% of the input radioactiv ity, as determined byWhatman DE81 fi l ter b inding.Isolation of RNAAfter in v itro transcription, the lysed nuclei were diluted with l-3 miof 0.25 M sodium acetate, 0.05 M Tris-HCI (pH 7.5). The lysate wasextracted twice with pheno l and once with chloroform and treatedwith DNAase as described previously (Zaug and Cech, 1980).Preparation of the Splic ing IntermediateRNA synthesized in isolated nuclei under the low salt condit ions andpurif ied as described above was ethanol-precipitated. The RNA from-2 X IO nu clei was redissolved in 0.05 ml of 0.05 M Tris-HCI (pH

7.5). It was then denatured in formamide and sedimented in a dena-turing sucrose gradient essentially as described by Anderson et al.(1974). We used 12 ml gradients containing 5%-20% sucrose in60% formamide , which were centr ifuged for 11.5 hr at 36,000 rpm,25OC in a Beckman SW41 rotor. Fractions were collected, and asmall aliquo t of each was spotted on a DE81 fi l ter to quantitate thelabele d RNA. The bulk of the RNA synthesized in v itro formed a broadpeak at 5s to 16S, and was discarded. The approximately 25% ofthe RNA that sedimented at 216s was collected by ethano l precipi-tation an d used as the splic ing intermediate .Excis ion of the IVS from the Splic ing intermedia teThe standard IVS-excis ion reaction involved incubation of the purif iedspl icing intermediate in 10 ~1 of 100 mM (NH&S04, 5 mM MgC12, 30mM Tris-HCI (pH 7.5), 0 .2 mM GTP for 30 min at 30C. Prior to beingdiv ided into 10 ~1 samples, the splic ing intermediate was mixed withall of the reaction components except one, either GTP or the com-ponent to be varied. The reactions were stopped on ice by theaddit ion of 20 mM EDTA. In the experiments in which the concentra-tion of one cofactor was varied, tha t component was added back,after the reactions were stopped, in amounts to equalize its concen-tration in all reaction tubes. Th e RNA was then ethanol-precipitated,and the precipitates were washed, dried and resuspended in 0.05 MTris-HCI (pH 7.5) before gel electrophoresis.Gel Electrophoresis of RNAUnless stated otherwise, s lab gels contained 4% polyacrylamide and8 M urea. RNA in urea-containing sample buffer was heated for 5 minat 65C prior to loadin g on the gel. The electrophoresis buffercontained 89 mM Tris-HCI, 89 mM boric acid, 2.5 mM EDTA (pH8.3). Electrophoresis was carried out for 2 hr at 35 mA constantcurrent at room tempe rature, or in an oven at 65OC to ensure completedenaturation. After soaking out the urea, we routinely dried the gelsand autoradiograph ed them overnight a t -70C using Kodak XRP-5or XAR-5 x-ray f i lm and a DuPon t Cronex intensify ing screen. Den-sitometry of x-ray f i lms was performed at 480 n m with the gel scanneraccessory of the Cary 219 spectrophotometer. Peak areas weredetermined with a Numonics electronic planimete r.End Analysis of RNARNAase T2 digestions of IVS RNA were carried out for I hr at 37Cin 10 pl of 0.01 M Tris-HCI. 0.001 M EDTA (pH 7.5) with 5 U enzyme.Nuclease Pl digestions were done as for T2, except at 6OC with 2U enzyme. Bacterial alkaline phosphatase incubations were done in10 ~1 of 0.01 M Tris-HCI (pH 8.0) for 1 hr at 37C with 100 U enzyme.Reaction mixtures were fractionated by high-voltage paper electro-phoresis on Whatman 3MM paper (Barrell, 1971). The samples werespotted 10 cm from the end of a piece of 3MM paper and allow ed todry completely. A small spot of dye mixture containing 1% each ofacid fuchsin. methyl o range and xylene cyanol was put on each s ideof the paper. Electrophoresis was carried out at 2500 V in 5% aceticacid, 0.3% pyridine, 1 mM EDTA (pH 3.5) unti l the xylene cyanol hadmigrated 10 cm from the origin.RNA Sequence AnalysisRNA was sequenced according to the procedures of Donis-Keller etal. (1977) and Donis-Keller (1980). with s light modifications. End-labeled IVS RNA in a solut ion conta in ing 250,ug/m l tRNA was par t ia llydigested with RNAase Tl or U2 at pH 3.5 for 15 min at 5OC. Enzymeto substrate ratios (units per microgram of tRNA) were 2 X 1 O-, 4X 1O-3 and 4 x 10e4 for T l and 2 X lo- * and 2 X 10m3 for U2.Partial digestions with RNAase Phy M (Donis-Keller. 1980) were doneat pH 5.0 with enzyme to substrate ratios of 4 x 1 O- and 8 x 1 O-.Partial alka line hydrolysis at pH 9.0 was carried out in sealed s ilatedcapil lary tubes at 90C for either 45 or 60 min. Sample s were loa dedon a preelectrophoresed 0.4 mm thick 20% polyacrylamide, 7 M ureagel and run at 30 W (1200-l 600 V) for 3 to 4 hr.Protease TreatmentsThe isolated unspliced pre-rRNA was incubated in the presence of10 mg/ml pronase (Sigma) or 0.4 mg/m l proteinase K (EM Biochem-

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icals) in a buffer containing 50 mM Tris-HCI (pH 7.51, 1 mM EDTAand 0.2% SDS (buffer A ). The incubations were performed in 10 ~1reaction mixtures at 37C for 60 min. Incubation of the precursor inbuffer alone constituted the control. Immediate ly after the proteasetreatments, the reactions were adjusted to splic ing condit ions on iceby the addit ion of a concentrated stock solution of splic ing buffer (1 Xbuffer: 10 mM MgCI., 100 mM (NH&S04 . 30 mM Tris-HCI [pH 7.51and 0.1 mM GTP) an d incubated at 30C for 30 min. (The proteasewas not removed prior to the splic ing reaction, s ince the splic ingactiv ity appears to be lost or inactivated by chloroform extraction atthis point.) In addit ion , the above protease treatments were performedafter the splic ing intermedia te was boiled for 5 min in buffer A. Thereactions were then adjusted to 5 mg/ml pronase or 0.2 mg/m lproteinase K and incubated for 1 hr at 37C in 90 pl volumes. Thesplic ing reaction was performed without the removal of the proteaseas described above (except for the larger volume). We assayed theproteases used in these treatments using known quantit ies of 14C-labele d proteins (Bethesda Research Laboratories). Under the con-dit ions specif ied above, pronasa routinely digested 82% of a 1 mgsample of a C-labeled protein-bovine serum album in mixture, andproteinase K digested 93% of the protein sam ple, as determined bytr ichloroacetic acid solubil ity .AcknowledgmentsWe thank Nancy Kan and Nanni D in for sharing unpublishe d DNAsequences; Norm Pace for advice on RNA sequencing; and NicholasCozzarell i for a helpful discussion. This work was supported by grantsfrom the Nation al Institutes of Health; the Council on Research andCreative Work of the University of Colorado; and the Biome dicalResearch Support Grant Program, Div is ion of Research Resources,Nation al Institutes of Health. T. R. C. was supported by an NIHResearch Career Developme nt Award.

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