Proc. Natl. Acad. Sci. USAVol. 89, pp. 5482-5486, June 1992Biochemistry

An ATP-independent U2 small nuclear ribonucleoproteinparticle/precursor mRNA complex requires both splicesites and the polypyrimidine tract

(precursor mRNA splicing/spliceosome/prespliceosome)

SHARON F. JAMISON*t AND MARIANO A. GARCIA-BLANCO*tt§*Section of Cell Growth, Regulation and Oncogenesis, tDepartment of Microbiology and Immunology and tDepartment of Medicine, Duke University MedicalCenter, Durham, NC 27710

Communicated by Phillip A. Sharp, February 7, 1992

ABSTRACT A complex is formed upon incubation of aprecursor mRNA (pre-mRNA) with HeLa cell nuclear extractin the absence of added ATP (-ATP complex). Pre-mRNAswith mutations in the 5' splice site, the 3' splice site, or thepolypyrimidine tract did not form this complex. Once formed,the -ATP complex was stable to competition by excess pre-mRNA. The complex was shown to contain the U2 smallnuclear ribonucleoprotein particle (snRNP) and was distinctfrom the previously described U2 snRNP/pre-mRNA complex,the prespliceosome. These complexes have different electro-phoretic mobilities, ATP requirements, and sensitivities tomutations of the 5' splice site. Although U1 snRNP was notfound in the -ATP complex, a requirement for the U1 snRNPwas suggested by immunodepletion experiments. The possibleimplications for the study of spliceosome formation are dis-cussed.

The splicing of precursors to messenger RNAs (pre-mRNAs)has been shown to occur in the spliceosome (1-3). Thespliceosome is a macromolecular assembly of pre-mRNA,U2, U4/6, and U5 small nuclear ribonucleoprotein particles(snRNPs, referred to collectively as U snRNPs, for uridine-rich) (4-6), and a set of proteins (7-13). There is controversyabout whether or not U1 snRNP is a stable component of thespliceosome during all the steps of splicing (4-6, 14). U1, U2,and U4/U6 snRNPs and protein factors have been shown tobe required for splicing (15-20). The late steps in the splice-osome formation pathway are highly conserved betweenbaker's yeast (Saccharomyces cerevisiae) and humans. Thisreflects in part the conservation of cis-acting elements inintrons of yeast and humans. These conserved sequences atthe 5' splice site and the 3' splice site define the boundariesof the introns.There are, however, remarkable differences in other in-

tronic cis-acting elements. In yeast, the branch-point se-quence is the highly conserved UACUAAC. In most animalintrons this conservation of sequence is not observed (19). Inanimal introns a polypyrimidine tract is conserved immedi-ately downstream ofthe branch-point sequence and is usuallylocated a few nucleotides upstream of the 3' splice siteconsensus sequence (19). In yeast an intronic polypyrimidinetract is not conserved. The branch-point sequence and thepolypyrimidine tract are involved in very early recognitionevents in the splicing pathway in yeast and animal introns,respectively (refs. 10, 14, and 20; R. Roscigno and M.A.G.-B., unpublished results). Therefore, the very early steps inspliceosome formation may be different in yeast and humans.A U1 snRNP/pre-mRNA splicing commitment complex is

formed in yeast nuclear extracts in the absence of exoge-

nously added ATP (14, 20). An early mammalian complex,the E complex, has been observed in the absence of exoge-nous ATP (13). The biochemical composition of this gradient-purified E complex remains to be established; however, thedata suggest that Ul snRNP is a component (13). Themammalian spliceosome formation pathway has been as-sumed to start with the recognition of the 5' splice site by theUl snRNP (21, 22). The second step has been described asthe ATP-dependent binding of U2 snRNP to the branch siteto form the prespliceosome (complex A) (1, 3, 5, 6, 14, 20, 23).This step has been shown to require U2 snRNP and severalprotein factors (24-26). In mammalian cells the formation ofthis complex requires a polypyrimidine tract and usually a 3'splice site (3, 10, 27). The prespliceosome and a U4/6/5multi-snRNP complex are assumed to come together, form-ing the spliceosome (complex B) (5, 28-30). Recent datashow that gradient-purified prespliceosomes can be con-verted to functional spliceosomes (unpublished results).

In this manuscript we present evidence for the formation ofan ATP-independent U2 snRNP/pre-mRNA complex inHeLa nuclear extract. The characterization of this complexmay provide insights into the mechanism of formation of theprespliceosome.

MATERIALS AND METHODSPlasmids and RNAs. pPIP3, pPIP3py5, and pPIP3py6 were

all described previously (10). (PIP indicates precursor inpieces; py, polypyrimidine tract.) pPIP4 was constructed byinserting the complementary oligonucleotides 5'-GGA-CAAACTCTTCGCGGTCTTTGCATG-3' and 5'-CAAA-GACCGCGAAGAGTTTGTCCTGCA-3' into pPIP3 linear-ized by Pst I and Sph I. The complete sequence of the pPIP4insert will be presented elsewhere (R. Roscigno and M.A.G.-B., unpublished results). pPIP4.2, pPIP4.3, and pPIP4.4 werecloned by replacing the Kpn I-BamHI fragment ofpPIP4 withmutant Kpn I-BamHI fragments derived from oligonucleo-tides (see Table 1 for sequences of mutant cis-acting ele-ments). The sequence of all plasmid inserts was determinedby dideoxy sequencing using Sequenase 2.0 (United StatesBiochemical) (31).

All RNAs were synthesized as described before (10, 32),except RNAs were labeled with [a-32P]UTP at a final specificactivity of 300 Ci/mmol in the labeling mix (1 Ci = 37 GBq).All RNAs were transcribed with T7 RNA polymerase (NewEngland Biolabs) from templates linearized with HindIII.

Nuclear Extracts and Splicing Reactions. Nuclear extractswere prepared by the method described by Dignam et al. (33).

Abbreviations: pre-mRNAs, precursors to messenger RNAs; sn-RNPs, small nuclear ribonucleoprotein particles; snRNAs, RNAcomponents of snRNPs.§To whom reprint requests should be addressed.

5482

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Proc. Natl. Acad. Sci. USA 89 (1992) 5483

Splicing reactions were as described previously (10). Finalconcentrations were 33% (vol/vol) nuclear extract, 62 mMKC1, 2mM MgCl2, 1 mM ATP, and 5 mM creatine phosphate.Reaction mixtures were incubated at 30'C for 10 min unlessotherwise indicated. Mixtures were placed on ice and sup-plemented with heparin (Sigma catalog no. H-3125) to a finalconcentration of 0.5 mg/ml and incubated at 30'C for 5 min.For the experiments illustrated in Fig. 3 D and E the heparinused was low molecular weight, -4000-6000 (Sigma catalogno. H-5640). Splicing reaction mixtures were usually incu-bated with 50,000 cpm of labeled RNAs or with 1 ug ofunlabeled RNAs for Northern blot assays. Splicing reactionproducts (5-7 1.d) were loaded on nondenaturing polyacryl-amide gels and electrophoresis was at 200 V (12.5 V/cm) atroom temperature for 3 hr (34). Gels were dried at 800C underreduced pressure for 1 hr. Splicing complexes were visual-ized by autoradiography with Kodak XAR-5 film and oneintensifying screen. Transfer of gels to GeneScreen mem-branes (DuPont) and the Northern blot procedure were asdescribed (32). The riboprobes for detection of the RNAcomponents of snRNPs (snRNAs) were as described byKonarska and Sharp (5). A riboprobe for pre-mRNA wasderived from EcoRI-digested pPIP2DNA (a PIP4 derivative),transcribed with T3 RNA polymerase (Stratagene).

Antibodies, Antisera, and Immune Assays. The JM humanserum was obtained from a healthy volunteer and the AW andGA human sera were obtained from patients with systemiclupus erythematosus, a gift of Jack Keene (Duke UniversityMedical Center). The rabbit antiserum to human immunode-ficiency virus rev protein was a gift of Bryan Cullen (DukeUniversity Medical Center). The monoclonal antibody to UlsnRNP 70-kDa protein was a gift of Jack Keene. For theimmunodepletion experiments, antibodies were absorbed onprewashed staphylococcal protein A-Sepharose CL-4Bbeads (Pharmacia) at 4°C for 1 hr to overnight. The beadswere washed twice with chilled immunoprecipitation buffer 1(10 mM Tris HCl, pH 7.5/200 mM NaCl/2.5 mM MgCl2/0.5% Nonidet P-40) and twice with chilled phosphate-buffered saline. The beads were spun at 14,000 x g at roomtemperature for 20 sec and the supernatant fraction wasdiscarded. A bead pellet volume of 30 ,ul was incubated with20 ,ul of HeLa nuclear extract at 4°C for 1-2 hr with mixing.The beads were spun at 14,000 x g at room temperature for30 sec and 15 ,l of the supernatant fraction (immunodepletednuclear extract) was collected and used in splicing reactionsas described above.

RESULTSA Stable Complex Is Formed in the Absence of Exogenously

Added ATP. PIP4 RNA, a pre-mRNA derived from theadenovirus-2 leader sequences, formed splicing complexesupon incubation with HeLa cell nuclear extracts (bands Aand B in Fig. 1). The splicing complexes A and B contain U2snRNA, and U2, U4/6, and U5 snRNAs, respectively; thelatter complex is the spliceosome (data not shown). Theformation of splicing complexes A and B required the addi-tion of 1 mM ATP and 5 mM creatine phosphate (Fig. 1, lanes9-16). PIP4 RNA incubated with HeLa cell nuclear extract inthe absence of added ATP formed a complex termed the-ATP complex, which migrated slower than complex A innondenaturing gels (* in Fig. 1, lanes 1-8). The -ATPcomplex also formed after extensive depletion of ATP in theextracts by preincubation, documented by depletion of[a-32P]ATP (data not shown). Moreover, addition of ATPresulted in a dramatic disappearance of the complex.The formation of this complex was MgCl2 concentration

dependent, with maximal levels of the complex seen at 2.0mM MgCI2 (Fig. 1, lanes 1-8). The -ATP complex, onceformed, was stable. The level of -ATP complex was un-

[MgCI2]mM 00411.212.012.813.614.415.2 0 |0o4|122.0|2.813.6|4A.5.2|ATP _ +

ori - A .,= s,*-B

-A

OH

l

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 1. MgCl2 concentration dependence of splicing complexformation. Splicing reaction mixtures were incubated with PIP4RNA and no added ATP (lanes 1-8) or with ATP and creatinephosphate added to 1 mM and 5 mM, respectively (lanes 9-16).Concentration of added MgCl2 is indicated above each lane. Splicingcomplexes were resolved by nondenaturing polyacrylamide gel elec-trophoresis and visualized by autoradiography. Heterogeneous com-plexes are indicated by H. The prespliceosome and the spliceosomeare indicated by A and B, respectively. The complex formed in theabsence ofATP (the -ATP complex) is indicated by *. The origin isindicated by ori.

changed after 10, 20, and 30 min of incubation in the presenceof >1000-fold molar excess of competitor pre-mRNA (datanot shown).The -ATP Complex Is pre-mRNA Specific. To investigate

the specificity of the -ATP complex we utilized mutants ofthe PIP4 RNA. The 5' splice site mutant PIP4.2 RNA formedlow levels of complex B and of the -ATP complex (40% ofwild type), whereas the formation of complex A was onlyslightly reduced (83% of wild type) (Fig. 2). The level of the-ATP complex, given in percent of wild type, was deter-mined by scanning the autoradiogram shown in Fig. 2; this

RN PIP1 PIP PIP PIP PIPRNA 4 4.2 3 3py513py6]A wt A5ss wt A3ss ApyATP - +-+ - I+ - a+- +

ori-

B- IIIi

on-~ ~

FIG. 2. -ATP complex formation is dependent on sequences atthe 5' splice site, the 3' splice site, and the polypyrimidine tract.Splicing reaction mixtures were incubated with wild type (wt)pre-mRNAs PIP4 (lanes 1 and 2) or PIP3 (lanes 5 and 6) or withmutant pre-mRNAs PIP4.2 (lanes 3 and 4), PIP3py5 (lanes 7 and 8),or PIP3py6 (lanes 9 and 10). The cis-acting elements mutated in theseRNAs are indicated in the figure: A5'ss represents a mutation in the5' splice site, A3'ss in the 3' splice site, and Apy in the polypyrimidinetract. The splicing reaction mixtures were incubated and splicingcomplexes were visualized as in Fig. 1. Symbols are as in Fig. 1.

.0L*

Biochemistry: Jamison and Garcia-Blanco

5484 Biochemistry: Jamison and Garcia-Blanco

Table 1. Effect of mutations on formation of splicing complexes

Polypyrimidine Formation of complexRNA 5'ss tract 3'ss A * A B

PIP4 GGG-GUGAGU........CCCUUUUUUUUCCACAG-CTC wt ++ ++ ++PIP4.2 GGG-AUGAU...........CCCUUUUUUUUCCACAG-CTC 5'ss +/- + -PIP4.3 GGG-AGAAU...........CCCUUUUUUUUCCACAG-CTC 5'ss +/- + -PIP4.4 GGG-GAGAAU...........CCCUUUUUUUUCCACAG-CTC 5'ss +/- + -PIP3py5 GGG-GUGAGU..........CCCUUUUUUUUCCGGAG-CTC 3'ss - - -PIP3py6 GGG-GUGAGU. CCCAUU&UUAUCCACAG-CTC py +/- +/- -

The PIP RNAs are identical transcripts except for the nucleotides in boldface type. The 5' splice site (5'ss) and the 3'splice site (3'ss) are indicated by hyphens. The cis-acting element that is changed by the mutations is indicated by thefollowing symbols in the A column: functional pre-mRNA or wild-type phenotype (wt); 5' splice site mutants (5'ss); 3' splicesite mutant (3'ss); polypyrimidine tract mutant (py). Formation of the -ATP complex (*), the A complex, or the B complexis indicated in the appropriate column: ++, no inhibitions; +, formation inhibited <50%; +/-, formation inhibited >70%o;-, formation inhibited >90%b.

resulted in an overestimate of this level. Other 5' splice sitemutants PIP4.3 and PIP4.4 yielded the same result: that is,the effect on the -ATP complex was more than 2-fold that of

the effect on complex A (see Table 1). The functionalpre-mRNA PIP3 formed all three complexes, whereas adouble point mutant in the 3' splice site, PIP3py5, did not

APre-mRNA Probe

RNA - PIP3 3py5A _ w t A3SS'ATP - + 1+ 94i

ori-

.

BU2 snRNA Probe

RNA I- PIP3 PIP3py5

A - wt A3SSSATP - + + < 4or' --

-*

-A

snRNA Probe

RNA P- P2 piP3pyS5jA .31 £xSS

ATP - *t__..o......_n.. ...

B

snRNP- IVA A

1 2 3 4 5 6

EDPre-mRNA Probe

-H

1 2 3 4 5 6

U2 snRNA Probe

RNA - wtATP -

ori--- .w

U2snRNP-

IH

F ; hcNh I n., NsnRNz \ B. andl i i SplICIn 1 IllI;ith un!claeledPIP!iRNA,:! ' 1icI'pn> RN

oi noU RNA (lneIt,A 1 iid >phiCMn cI( c tI I!! c Y

-Upplemnitnted cvl h rl N P l in nfth4.a d 6) oru nd suLpplemnLcrntcc Iot h I".II'i ei.oilk l3 icL'

_ ines i. and Splic. izll c }Il lC\C'2 "C -01\tuiingh gel tlecr ph and!rl ]; t'cjl'_d oot :IciccLTohrc.nd ..tc½ trOtc½ i

--ninhjtlrn .The cnlt'0 inc cv- ':i rmirohcJ t1 f il .. IIkv;lIP pitcnmRNA,Nc1l. ' nR \1,nRNAN ( I(1) and /-1 Splicir C 1T n i n. cii K i Icnith cmL bh~clcc 'tIPI RN.N, "1EiIIFtl''n;,X ; ;''~klth UnlabeleI 1F[niir viir- 11\tEi.kNAikneIF."rhtll~PkRNAN nLc Spllii Ic I !I Ll C iO IIi c 11Tvcc th NATP or .rulini nho~pnl id'c' p'1csolved 6% noindcnounnii mmCl clccp T 1c i(. tni ccS i r Ii ()i i nnihiu'n.t c i.cii Ito htparinn ci Accci. chclIc isplicing cc !nip l.'c I.PcXiliT L.

specifieCIC fOr PII P pic^ R N'1-nriAit .1

cndogenouc1 1 2' ( nLiin I 1 -i.. K \ A tAnlNanclP 1.id I . _nRN

UlsnRNP-1

Rt-Twt 1|s3RNA Ssw

ATP - J

ori- 00t-

.,

. 3x.I

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 5485

form any of these complexes (Fig. 2 and Table 1). PIP3 andPIP4 RNAs differ only in the size of the downstream exon.A triple point mutant in the polypyrimidine tract, PIP3py6,was also severely impaired in the formation of all threecomplexes (Fig. 2 and Table 1). These experiments wereconfirmed with competition assays (data not shown).

Biochemical Composition of the -ATP Complex. To char-acterize the components of the -ATP complex we employedtwo techniques: antiserum-mediated supershifting andNorthern blotting.The mobility ofthe -ATP complex was minimally changed

by incubation with normal human serum JM. The mobility ofthe -ATP complex was markedly retarded (supershifted) byincubation with human serum AW, which has a high titeragainst Sm determinants on snRNPs (data not shown). Splic-ing complexes A and B showed the same behavior, beingsupershifted with the anti-Sm human serum but not with thenormal human serum JM. These experiments suggested thepresence of a U snRNP or a U snRNP component in the-ATP complex.The presence of U snRNAs comigrating with splicing

complexes, as detected by Northern blots, has stronglysuggested the presence of the U2, U5, and U4/U6 snRNPs inthese complexes (5). Other methods have, in part, validatedthis conclusion (4, 14, 35). We used Northern analysis toidentify U snRNAs in the -ATP complex. Unlabeled PIP3RNA was incubated with the nuclear extract and electropho-resed on nondenaturing polyacrylamide gels. The splicingcomplexes were transferred to a GeneScreen nylon mem-brane. The membrane was probed with labeled RNAs com-plementary to the PIP3 RNA, and to several U snRNAs. Theanti-PIP3 RNA probe clearly showed that unlabeled PIP3RNA was incorporated into the -ATP complex and in thepresence of added ATP into the A and B complexes (Fig. 3Alanes 3 and 4). PIP3py5 RNA, a 3' splice site mutant, did notget incorporated into the -ATP or the A and B complexes(lanes 5 and 6). This RNA, as well as PIP3 RNA, formednonspecific H complexes (lanes 3-6). As expected, no hy-bridization was observed when pre-mRNA was not added tothe nuclear extracts during the incubations (lanes 1 and 2).The same membrane was stripped and reprobed with U

snRNA-specific probes. A U2-specific probe detected U2snRNA migrating with the -ATP complex (* in Fig. 3B, lane3). As expected, this complex was seen only with PIP3 RNAand not upon incubation with mutant RNA or without RNA(lanes 1 and 5). This probe also detected the endogenous U2snRNP in reaction mixtures where pre-mRNA was not added(lanes 1 and 2). The -ATP complex migrated immediatelyadjacent to the endogenous U2 snRNP. The U2-specificprobe also detected U2 snRNA that comigrated with the Aand B complexes, as has been reported before (32) (lane 4).A U1 snRNA-specific probe did not detect U1 snRNAcomigrating with any of the complexes formed by PIP3 orPIP3py5 RNAs (Fig. 3C). This probe detected the endoge-nous U1 snRNA that migrates as a broad band of complexes.These results argued that U2 snRNP but not Ul snRNP waspresent in the electrophoretically resolved -ATP complex.

This experiment was repeated with a different type ofheparin (see Materials and Methods) that afforded betterseparation of the -ATP complex from the endogenous U2snRNP. The use of this heparin resulted in the formation oftwo complexes in the absence of ATP. Pre-mRNA- and U2snRNP-specific probes clearly show that the U2 snRNAexactly comigrates with the -ATP complexes that form withPIP3 but not with the 3' splice site mutant PIP3py5 (Fig. 3 Dand E). A U1 snRNA probe did not detect U1 snRNA underthese conditions. The mobilities of splicing complexesformed in the presence of ATP were also changed when thisheparin was used (data not shown).

Immunodepletion ofU1 snRNP Inhibits the Formation of the-ATP Complex. The requirement for U1 snRNP for theformation of the splicing complexes was evaluated by immu-nodepletion. HeLa nuclear extracts were incubated withprotein A-Sepharose beads loaded with monoclonal antibod-ies or antisera against specific antigens. Immunodepletionwith human serum AW against the Sm determinant or withhuman serum GA against U1 and U2 snRNP proteins resultedin undetectable levels of -ATP, A, and B complexes (datanot shown). Immunodepletion with a monoclonal antibody tothe U1 snRNP 70-kDa protein resulted in a dramatic decreasein the level of -ATP complex (Fig. 4, lane 5). This effect wasnot seen with control antisera. There was also a markedreduction in the level of complex B (lane 6), as previouslyreported by Barabino et al. (17). These experiments stronglysuggest that U1 snRNP is required for the formation of the-ATP complex. The effect on complex A was small, con-trary to that observed by others (17) and by us, usingoligonucleotide depletion of U1 snRNP (unpublished re-sults). This oligonucleotide depletion confirmed the require-ment for U1 snRNP in the formation of the -ATP complex.

DISCUSSIONWe describe here a complex formed when PIP pre-mRNAsare incubated in HeLa cell nuclear extracts. The formation ofthis complex did not require the presence of added ATP. This-ATP complex could be defined as a splicing-specific com-plex, given the strong negative effect noted with mutants incis-acting elements required for splicing. The biochemicalcomposition of this complex was not expected. Antibodysupershift experiments indicated that the -ATP complexincluded a U snRNP. Northern blots showed that U2 snRNAwas present in the -ATP complex, but U1 snRNA was notdetected in the complex. Finally, we showed that U1 snRNPwas probably required to form this -ATP complex. Thedifference in electrophoretic mobility suggested that the

Ab - R.S.IRNP lJMISpecificity_ a rev1k70D nl

ATP _1- + - +1- +1- +1- +

-B

-A

-H

1 2 3 4 5 6 7 8 9 10

FIG. 4. Formation of the -ATP complex requires the Ul snRNP.Splicing reaction mixtures were incubated with HeLa nuclear extract(lanes 1, 2, 7, and 8), with HeLa nuclear extract that had beenimmunodepleted of U1 snRNP with a monoclonal antibody to the U1snRNP 70-kDa protein (lanes 5 and 6), or with a nuclear extractmock-immunodepleted by using control sera: rabbit anti-rev protein(lanes 3 and 4) or normal human serum (lanes 9 and 10). Splicingreaction mixtures were incubated and splicing complexes werevisualized as in Fig. 1. Symbols are as in Fig. 1.

Biochemistry: Jamison and Garcia-Blanco

5486 Biochemistry: Jamison and Garcia-Blanco

-ATP complex was different from the prespliceosome (com-plex A). The differential effect of addition of ATP and thedifference in sensitivity to 5' splice site mutations confirmedthat the -ATP complex was not the A complex.The association of U2 snRNP with pre-mRNA in the

absence of ATP was surprising. The -ATP complex mayarise from a limited pool of activated U2 snRNP alreadypresent in the extracts. This is unlikely, however, becausethe -ATP complex is not readily observed with other func-tional pre-mRNAs tested. A rabbit B3-globin pre-mRNA didnot form a -ATP complex (data not shown). It is more likelythat the interaction of the PIP pre-mRNAs with U1 snRNP isflexible enough to permit the entry of the U2 snRNP in theabsence of added ATP. A similar ATP-independent U2snRNP complex has been observed to form with pre-mRNAsin yeast extracts containing the U1-4u mutant U1 snRNA (M.Rosbash, personal communication). Our favored hypothesisis that the -ATP complex is a U1 snRNP/U2 snRNP/pre-mRNA complex in solution and the U1 snRNP is lost duringelectrophoresis. The structure of this complex may be similarto transient intermediates formed during the ATP-dependentformation of the prespliceosome from a U1 snRNP/pre-mRNA complex.The -ATP complex is distinct from the previously de-

scribed complex A by the following criteria: (i) increasedsensitivity to mutations of the 5' splice site, (ii) increasedsensitivity to depletion of U1 snRNP, (iii) lack of a require-ment for addition of ATP, and (iv) differential migration onnondenaturing polyacrylamide gels and glycerol gradients.The study of the -ATP complex may provide insights intothe molecular interactions between the U1 snRNP and the U2snRNP early in the splicing pathway.

Note. A recent paper by Michaud and Reed (36) describes a complexE that forms in the absence of added ATP. This HeLa cell complexdetected by gel filtration contains U1 and U2 snRNPs, and it isformed with an adenovirus-derived pre-mRNA but not with a human,3-globin. The peak fraction of the complex E was shown to havecommitment activity. We believe the -ATP complex that we de-scribed above is equivalent to complex E. We have shown thatalthough the -ATP complex peak fractions have commitment ac-tivity, this activity does not copurify with this complex. The purifi-cation of commitment activity relative to complex E was not ad-dressed by Michaud and Reed.

We thank J. Keene, E. Holmes, R. Roscigno, and R. Colvin forcritical reading of this manuscript and for many helpful discussions.We also thank P. Sharp, M. Konarska, M. Brown, and M. Moore formany helpful discussions and M. Rosbash for communicating databefore publication. We thank P. Spears and M. Loomis for help inpreparation of the manuscript. This work was supported by start-upfunds from the Duke University Medical Center and grants from theAmerican Cancer Society and the National Institutes of Health toM.A.G.-B.

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