NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com 929

letters to nature

.................................................................Stimulatory effect of splicing factorson transcriptional elongationYick W. Fong & Qiang Zhou

Department of Molecular and Cell Biology, University of California at Berkeley,Berkeley, California 94720-3206, USA..............................................................................................................................................

Transcription and pre-mRNA splicing are tightly coupled geneexpression events in eukaryotic cells1,2. An interaction between thecarboxy-terminal domain of the largest subunit of RNA polymer-ase (Pol) II and components of the splicing machinery is postu-lated to mediate this coupling3±5. Here, we show that splicingfactors function directly to promote transcriptional elongation,demonstrating that transcription is more intimately coupled tosplicing than previously thought. The spliceosomal U smallnuclear ribonucleoproteins (snRNPs) interact with human tran-scription elongation factor TAT-SF1 (refs 6±9) and stronglystimulate polymerase elongation when directed to an intron-free human immunode®ciency virus-1 (HIV-1) template. Thiseffect is likely to be mediated through the binding of TAT-SF1 toelongation factor P-TEFb10, a proposed component of the tran-scription elongation complex11,12. Inclusion of splicing signals inthe nascent transcript further stimulates transcription, support-ing the notion that the recruitment of U snRNPs near theelongating polymerase is important for transcription. Becausethe TAT-SF1±U snRNP complex also stimulates splicing in vitro, itmay serve as a dual-function factor to couple transcription andsplicing and to facilitate their reciprocal activation.

The Pol II C-terminal domain (CTD) is hyperphosphorylatedduring the transcription cycle at a time coincident with processivepolymerase elongation13. Phosphorylation of the CTD duringelongation is carried out primarily by P-TEFb, a heterodimer ofCDK9 and cyclin T1 (CYCT1)10. P-TEFb is also a cellular cofactorfor the HIV-1 TAT protein, and the TAT±P-TEFb complex stimu-lates HIV-1 transcriptional elongation by interacting with the TARRNA structure located at the 59 end of the nascent viral tran-script14,15. Although the cyclin box located in the amino-terminalhalf of CYCT1 is essential as it contacts CDK9, TAT and TAR16, theCYCT1 C-terminal half has also been shown to contribute signi®-cantly to both basal and TAT-stimulated HIV-1 transcription17,18.

The importance of the CYCT1 C-terminal domain prompted usto identify and analyse transcription factors that may associate withthis domain. We therefore incubated nuclear extract of HeLa cellswith immobilized GST or GST±CYCT1-C, which contained aCYCT1 C-terminal fragment (amino acids 402±701; ref. 18).Compared with the GST-depleted extract, extract depleted withGST±CYCT1-C showed a signi®cant decrease (about ninefold onaverage) in basal as well as TAT-dependent HIV-1 transcriptionfrom both templates: pHIV+TAR-G400, which contained the wild-type TAR element; and pHIVDTAR-G100, with a mutant TAR19

(Fig. 1a). Because the level of TAT activation (about eightfold)was largely unaffected by the depletion, CYCT1-C probablyremoved general transcription activity.

In nuclear extract of HeLa cells, CYCT1-C has been shown tointeract with Pol II and TAT-SF118, one or both of which maycontribute to the activity depleted by CYCT1-C. TAT-SF1 has beenidenti®ed as a TAT cofactor as well as a general transcriptionelongation factor6±9. Because it is highly enriched in a partiallypuri®ed Q-Sepharose fraction of HeLa nuclear extract that containsa small portion of HeLa nuclear proteins (3±4%), including Pol II8,we examined whether this fraction can complement the CYCT1-C-depleted extract in transcription. HIV-1 transcription was restoredby the addition of the Q fraction pre-depleted with GST, but notwith GST±CYCT1-C (Fig. 1a), suggesting that the activity depleted

from HeLa nuclear extract by CYCT1-C also exists in the Q fraction.Western analysis revealed a quantitative removal of TAT-SF1 fromthe CYCT1-C-depleted Q fraction (Fig. 1b). However, CYCT1-Conly partially removed SPT5, an elongation factor reported to bindTAT-SF1 (ref. 6), and it removed very little Pol II or the TFIIFsubunit RAP30. Thus, Pol II was probably not responsible for theactivity depleted by CYCT1-C.

Rather, the CYCT1-C-depleted activity seemed to associate withTAT-SF1, because the immunoprecipitation of TAT-SF1 and itsassociated factors from the Q fraction with anti-TAT-SF1, but notwith preimmune antibodies, restored both basal and TAT-depen-dent HIV-1 transcription to a CYCT1-C-depleted nuclear extract(Fig. 1c). Because these reactions measured transcription from twoG-less cassettes (G-400 and G-100) located about 1 kilobase (kb)downstream of the HIV-1 promoter19, the anti-TAT-SF1 immuno-precipitates were postulated to transactivate at the level of elongation.To con®rm this, we performed an assay involving discontinuous

– –

– –– + – + – +

– – + + + +– + – + – + – +

c

IP from Q fraction:

IP from Q fraction:

Pre

-im

mun

eA

nti-

TAT-

SF1

Anti-

TAT-

SF1

Preim

mun

e

NE depletedwith CYCT1-C

NE depletedwith CYCT1-C

TAT:

1–82

1,241–1,495

+TAR-G400

∆TAR-G100

GST GST-CycT1-C

GST GST–CYCT1-C

Depleted NE:

Depleted Q:

TAT:

a

GST GST–

CYCT1-Cb

TARX+1 +955 +1,067

G-LESSpHIV∆TAR-G100 TATA

Sp1TATA TAR

+1 +955 +1,340pHIV+TAR-G400

G-LESSTranscription templates

d

∆TAR-G100

+TAR-G400

Anti-Pol II

Anti-SPT5

Anti-TAT-SF1

Anti-RAP30

Depleted Q:

Figure 1 The CYCT1 C-terminal domain interacts with a TAT-SF1-associated

transcription elongation activity. a, HeLa nuclear extract (NE) and the Q-Sepharose

fraction (Q) were depleted with immobilized GST or GST±CYCT1-C and then incubated

with templates pHIV+TAR-G400 and pHIVDTAR-G100 in reactions with or without Tat.

The RNase T1-resistant G-less transcripts derived from the two templates are indicated.

b, The levels of the indicated proteins in Q depleted with GST or GST±CYCT1-C were

analysed by western blotting. c, The Q fraction was subjected to immunoprecipitation with

preimmune or anti-TAT-SF1 antibody beads. The immunoprecipitates (IP) were analysed

in reactions containing the CYCT1-C-depleted NE as in a. d, A discontinuous hybridization

followed by RNase protection assay20 was performed to analyse the level of transcription

from template pHIV+TAR at two different distances from the initiation site. Reactions

contained the CYCT1-C-depleted NE and the indicated immunoprecipitates. Numbers

denote the 59 and 39 extent of the protected RNA fragments.

© 2001 Macmillan Magazines Ltd

hybridization followed by RNase protection20 to measure transcrip-tion at two different locations from the initiation site (Fig. 1d).Addition of the TAT-SF1 immunoprecipitates to the CYCT1-C-depleted extract only weakly stimulated the promoter-proximaltranscription (nucleotides 1±82), but enhanced the promoter-distal transcription (nucleotides 1,241±1,495) by 20-fold, revealinga predominant effect on elongation.

Sequence comparisons indicate that of all vertebrate proteins,human and mouse TAT-SF1 proteins are most homologous toCUS2 of Saccharomyces cerevisiae. The homology lies within thetwo atypical RNA recognition motifs (RRMs) and their ¯ankingregions located in the N-terminal half of TAT-SF1 (Fig. 2a). Theextensive acidic C-terminal domain in human TAT-SF1 is reducedto a 25-amino-acid segment in CUS2. CUS2 was identi®ed as asuppressor of a U2 snRNA mutation and shown to regulate thespliceosomal formation21,22. In splicing extracts, CUS2 associateswith U2 snRNA and splicing factor SF3a21.

The homology between TAT-SF1 and CUS2 suggests that TAT-SF1 may also bind splicing factors, which could be responsible forthe observed stimulatory effect on transcriptional elongation. Totest these hypotheses, a TAT-SF1 mutant containing a point muta-tion (Y136D) in the ®rst RRM was generated (Fig. 2a). A corre-sponding CUS2 mutation (Y48D) destroys the CUS2±U2 snRNAinteraction and abrogates CUS2 activity21. Because the extended C-terminal half of TAT-SF1 is absent in CUS2, a truncated TAT-SF1,termed DC (amino acids 1±380; Fig. 2a) was also generated. Flag-tagged wild-type and mutant TAT-SF1 and their associated proteins(TAT-SF1±Flag immunoprecipitates) were af®nity puri®ed fromthe nuclear extracts of transfected 293T cells and examined bywestern blotting. Consistent with CUS2 being a splicing factor,wild-type TAT-SF1±Flag interacted with the U1 snRNP-speci®cprotein U1 70K, U2-speci®c protein U2B0, and the common Smproteins B and B9 (Fig. 2b). The Y136D mutation destroyed all ofthese interactions, whereas the DC deletion showed no effect.Northern hybridization (Fig. 2c) further demonstrated that boththe TAT-SF1 immunoprecipitates isolated from the Q fraction andthe transfected wild-type TAT-SF1±Flag puri®ed from the nuclearextract associated with all ®ve spliceosomal U snRNAs. Again, theY136D mutation disrupted these interactions. Compared with wild-type TAT-SF1±Flag, DC interacted with less U5 and U4/U6. Thus,the TAT-SF1 RRM domain seemed to be primarily responsible forthe interactions of TAT-SF1 with the snRNPs, although the C-terminal acidic domain also contributed to some of the interactions.

In reactions containing the CYCT1-C-depleted HeLa nuclearextract, addition of the wild-type TAT-SF1±Flag immunopre-cipitate increased HIV-1 transcription (Fig. 2d). In contrast,transcription was slightly decreased by the DC preparation andsigni®cantly inhibited by the Y136D preparation, suggesting thatboth the RRM and the acidic C-terminal domains of TAT-SF1 arerequired for transcription. To explain these differences, the wild-type and mutant TAT-SF1±Flag immunoprecipitates were tested forinteracting with immobilized GST±CYCT1-C (Fig. 2e). Whereasboth the wild-type and Y136D preparations showed active binding,the DC complex failed, revealing a requirement for the TAT-SF1 Cterminus in contacting P-TEFb. P-TEFb was reported to associatewith the Pol II elongation complex along the HIV-1 templatethrough position +529 (refs 11, 12), which may recruit the TAT-SF1±snRNP complex to Pol II through the CYCT1±TAT-SF1interaction. Therefore, the lack of transactivation by DC could beattributed to its failure to associate with the elongating Pol IIthrough CYCT1, and/or its decreased binding to U5 and U4/U6snRNPs. Although Y136D did not associate with any snRNP, itinteracted with CYCT1-C even stronger than did the wild-typeprotein (Fig. 2e). This may prevent the recruitment of a functionalTAT-SF1±snRNP complex to Pol II and thus inhibit transcription.

These interpretations were based on the assumption that theTAT-SF1-associated splicing factors are capable of transactivation.To con®rm this, the immobilized TAT-SF1 immunoprecipitateswere treated with micrococcal nuclease and washed extensively toremove the disrupted snRNPs. Like Y136D, the material treatedwith micrococcal nuclease inhibited transcription mediated by theCYCT1-C-depleted extract (Fig. 3a), suggesting that TAT-SF1 aloneis incapable of transactivation.

Further proof of the transactivating activity of the TAT-SF1±snRNP complex came from two depletion studies. First, wedepleted HeLa nuclear extract with a monoclonal antibody(K121) recognizing the unique 2,2,7-trimethylguanosine (TMG)cap structure at the 59 end of all the spliceosomal U snRNAs exceptU6. This antibody has been used to af®nity purify U snRNPs fromHeLa extract23,24. Compared with the control anti-HA-depletedextract, the anti-TMG-depleted extract showed a decrease in HIV-1 transcription (Fig. 3b), which was reversed by the addition ofwild-type TAT-SF1±Flag immunoprecipitate. A reported directinteraction between yeast U2 snRNP and CUS2 (refs 21, 22) suggests

letters to nature

930 NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com

Acidic domain754

C420

RRM RRM FN1

RNA recognition motifs

WT TAT-SF1

Y136D

∆C380

F1

Y136D

F

RRM RRM2851

CUS2

∆C

Anti-Flag

Anti-U2B"

Anti-Sm

WT

Y13

6D

b TAT-SF1–Flag IP

Anti-U1 70K

Western blot:

U4

U5U6

U1

U2

∆C

∆C Y13

6DW

T

WT

Y13

6D

NE

Q fr

actio

n

TAT-SF1–Flag IP

IP fromQ fraction

Pre

imm

une

Ant

i-TAT

-SF1

c

NE depletedwith CYCT1-C

TAT-SF1–Flag IP: –

∆TAR-G100

+TAR-G400

e TAT-SF1–Flag IP

∆C WTY136D

Inpu

t

Inpu

t

Inpu

t

GST

GST

GSTCYC

T-C

CYC

T-C

GST pull-down andanti-Flag western blot

d

CYC

T-C

a

Figure 2 Both the N-terminal RRMs and the C-terminal acidic domain of TAT-SF1 are

required for transactivation. a, Domain structures of wild-type (WT) and mutant (Y136D

and DC) TAT-SF1 and the yeast homologue of TAT-SF1, CUS2. b, Flag-tagged wild-type

and mutant TAT-SF1 were af®nity puri®ed from the nuclear extracts of transfected 293T

cells and analysed for their associated snRNP proteins by western blotting with the

indicated antibodies. IP, immunoprecipitate. c, RNA recovered from the indicated sources

was analysed by northern hybridization with snRNA-speci®c riboprobes. d, Af®nity-

puri®ed wild-type and mutant TAT-SF1±Flag and their associated factors were analysed in

transcription reactions containing the CYCT1-C-depleted HeLa nuclear extract (NE) as in

Fig. 1a. Because TAT-SF1±snRNP affected general HIV-1 transcription, TAT was therefore

omitted from this and the subsequent experiments, although the same two-template

system was used for consistency. e, The indicated TAT-SF1±Flag immunoprecipitates

were incubated with immobilized GST and GST±CYCT1-C. The bound proteins and 38%

of the input materials were examined by anti-Flag western blotting.

© 2001 Macmillan Magazines Ltd

that a similar U2 snRNP±TAT-SF1 interaction may be required fortransactivation. U2 snRNP was therefore depleted from HeLanuclear extract by hybridization to an immobilized U2 antisenseRNA oligonucleotide25. Compared with a mock-depleted extract,the extract depleted with antisense RNA showed a decrease in HIV-1transcription, which was again rescued by the TAT-SF1±Flagimmunoprecipitates (Fig. 3b).

In a reciprocal experiment, U2 snRNP and its associated factorswere precipitated from the Q fraction using the U2 antisense RNAbeads. On elution with a displacement DNA oligonucleotide26, thefraction was found to contain TAT-SF1 (data not shown), andincreased transcription when added to the CYCT1-C-depletednuclear extract (Fig. 3c). That an activity capable of complementingthe CYCT1-C-depleted extract was isolated from the Q fractionusing either anti-TAT-SF1 antibodies or U2 antisense RNA arguesthat this activity resides in a single complex that minimally containsTAT-SF1 and U2 snRNP.

The TAT-SF1±snRNP complex also complemented HeLa nuclearextract treated with micrococcal nuclease to splice a b-globin pre-mRNA (Fig. 3d). The treatment degraded all major RNA species inthe extract, and abolished splicing27. Addition of the TAT-SF1immunoprecipitates isolated from the Q fraction restored splicingto the micrococcal-nuclease-treated extract. Thus, the TAT-SF1±snRNP complex seemed to function in both splicing andtranscription.

letters to nature

NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com 931

––

––+ +

b

Moc

kU2

AS

4 5 6

Moc

kU

2 ASPurified

from Q:

TAT-SF1 –Flag IP:

1 2 3

Anti-H

A

Anti-T

MG

c

10.

090.72 1

0.33 1.

6

1 3

Preim

mun

e

Anti-T

AT-S

F1

MN:

1 2 3 4

1.1 1

0.08 11

a

MNtreatmentM

ock

Pre

imm

une

Ant

i-TAT

-SF1

d

1 2 3 4M

NE depletedwith CYCT1-C

NE depletedwith CYCT1-C

IP from Q fraction:

IP from Q fraction:

HeLa NE:

+TAR-G400

+TAR-G400

+TAR-G400

∆TAR-G100

∆TAR-G100

∆TAR-G100

Relativeunits

Relativeunits

Relativeunits

NE depleted with:

–– + –– +

Figure 3 The TAT-SF1-associated U snRNPs are active in both transcription and splicing.

a, The immobilized preimmune or anti-TAT-SF1 immunoprecipitates were treated with

micrococcal nuclease (MN), washed extensively to remove the degraded snRNPs, and

tested in reactions as in Fig. 1a. Relative levels of transcription were averaged between

the two transcripts in each reaction and normalized against the value (set at 1) in lane 2.

b, Transcription reactions contained HeLa nuclear extract (NE) depleted with anti-HA or

anti-TMG monoclonal antibody (lanes 1±3), or NE mock depleted or depleted with the U2

antisense (AS) RNA beads (lanes 4±6). Af®nity-puri®ed TAT-SF1±Flag and its associated

factors were added to the indicated reactions. c, U2 snRNP and its associated factors

were puri®ed from the Q fraction using agarose beads without (mock) or with the

immobilized U2 antisense RNA oligonucleotide. The eluted fractions were analysed in

transcription reactions as in Fig. 1a. d, Mock- or MN-treated NE was incubated with b-

globin pre-mRNA in reactions containing the indicated immunoprecipitates. Icons indicate

splicing intermediates and products. Lane M shows a 1-kb ladder (Invitrogen).

Template: W M W W M M

Anti-Sm:

Anti-tubulin:

0.8-kbrunoff

0.8-kbrunoff 0.7-kb

runoff

0.5-kbrunoff

Lariatintermediate

1 2 3 4 5 6

W M W W

1' 2' 3' 4'Relative

units

Relativeunits

1 0.27 0.89 0.21 0.25 0.22

Template: W M W M5'ss3'ss Py-t

Bp

Ad2 β-globinb

1 2 3 4 5 6 7 8

TATA+1 +502 +838

Ad2 EIE

TATA+1 +502 +714

β-globin EIE

Internal control template:+1 +502

TATA

Transcription/splicing templates

1 0.34 0.25 0.19 0.7 0.15 1 0.23

a

Template W (Ad2):

Template W (β-globin):

– – –+ + – –– –+

– – +– – + –– +–

Figure 4 Splicing signals in nascent transcripts stimulate transcription. a, HeLa nuclear

extract, linearized template W or M, and anti-tubulin or anti-Sm antibody were incubated

in coupled transcription and splicing reactions, which produced ,0.8-kb runoff

transcripts. Lanes 19±49 were exposed eight times longer than lanes 1±4 to reveal the

lariat intermediate. Template W and M contained, respectively, the wild-type and mutant

(mutations at 59 and 39 splice sites and the polypyrimidine tract) Ad2 exon±intron±exon

(EIE) sequences inserted downstream of the HIV-1 promoter. b, All reactions contained

HeLa nuclear extract and two templates. The internal control template produced an intron-

free runoff transcript of ,0.5 kb. The second set of templates contained Ad2 or b-globin

EIE and produced intron-containing transcripts of ,0.8 or ,0.7 kb. Four indicated

templates carried individual mutations at the 59 (59ss) or 39 splice site (39ss), the

polypyrimidine tract (Py-t), or the branch point (Bp) in Ad2 EIE. Template M contained

multiple mutations as described in a. Relative levels of transcription were normalized

against the internal controls.

© 2001 Macmillan Magazines Ltd

We used intron-free templates in all transcription reactionsdescribed thus far. Recruitment of TAT-SF1±snRNP to these tem-plates is probably mediated by the interaction of TAT-SF1 withCYCT1±P-TEFb, which was shown to travel with the elongatingpolymerase11,12. For intron-containing templates, however, the pre-sence of splicing signals in the nascent transcript may attractadditional snRNPs near Pol II to further stimulate transcription.To test this hypothesis, we inserted a 336-base pair (bp) adenovirusDNA with wild-type exon±intron±exon sequences (pSPAd28)downstream of the HIV-1 promoter (Fig. 4b). This template waslinearized and a runoff transcript of about 0.8 kb was expected.Compared with a mutant template containing the mutated 59 and 39splice sites and the polypyrimidine tract, the wild-type templatedisplayed increased transcription (Fig. 4a, lanes 1 and 2). A longerexposure revealed a low level of splicing of only the wild-typetranscript, as evidenced by the generation of a splicing intermediate(lariat intermediate) (lanes 19 and 29). Inhibition of splicing by theanti-Sm antibody29 reduced transcription from the wild-type butnot the mutant template. Furthermore, individual mutations of the59 and 39 splice sites and the polypyrimidine tract, but not thebranch point adenosine, largely impaired the ability of the adeno-virus intron to stimulate transcription (Fig. 4b). Con®rming anintron-dependent stimulation process, a similar level of shorter(0.5-kb) transcripts was produced from an internal control templatelinearized before the exon±intron insert. Finally, an inserted b-globin intron also increased transcription, revealing the generalityof the phenomenon (Fig. 4b). On the basis of these results, wepropose that the transcriptionally active TAT-SF1±snRNP complexcan be recruited to the elongation complex through the binding ofTAT-SF1 to P-TEFb and the binding of snRNPs to the nascentsplicing substrate. Notably, only the later process seemed to besensitive to anti-Sm inhibition.

Although the major events in eukaryotic gene expression havetraditionally been analysed individually, recent evidence indicatesthat they are highly coupled inside the cell1,2. The couplingsdescribed thus far, however, have largely been unidirectional,involving one upstream event (for example, splicing) affectingone or more downstream events (for example, mRNA export anddecay). Our observations that the TAT-SF1-associated splicingfactors and the splicing signals in the nascent transcript promotetranscriptional elongation suggest that the coupling between tran-scription and splicing can be mutually bene®cial. As P-TEFb istravelling with the elongation complex11,12, the CYCT1±TAT-SF1interaction may recruit TAT-SF1±snRNP to the elongating poly-merase, increase the local concentration of splicing factors, andfacilitate the assembly of a spliceosome when splice sites emergefrom the polymerase. This mechanism, combined with the associa-tion of splicing factors with Pol II CTD3±5, may explain the highef®ciency of co-transcriptional splicing in vivo. Although splicingmay bene®t from the coupled transcription, our data suggest thatthe two processes can exhibit reciprocal synergism, providing anef®cient mechanism for their coordinated regulation in response tochanging physiological conditions. A stimulatory effect on geneexpression by the presence of introns in the transcription unit hasbeen observed in both yeast and mice30. Future experiments willshed light on the mechanism by which the TAT-SF1-associatedsplicing factors stimulate transcriptional elongation. M

MethodsDepletion of nuclear extracts

We incubated 0.5 mg of HeLa nuclear extracts in D buffer (20 mM HEPES buffer at pH 7.9,10% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.05% NP40, 1 mM dithiothreitol and 0.5 mMphenylmethyl sulphonyl ¯uoride) twice with 1 mg of immobilized GSTor GST±CYCT1-Cfor a total of 4 h at 4 8C to obtain the CYCT1-C-depleted extracts, or three times with anti-TMG monoclonal antibody coupled to protein G±Sepharose for a total of 3 h at 4 8C toobtain the anti-TMG-depleted extracts. Depletion of U2 snRNP was carried out asdescribed25 except that nuclear extracts in D buffer were used.

Af®nity puri®cation of TAT-SF1 complexes

Flag-tagged TAT-SF1 proteins were transiently expressed in 293T cells. Nuclear extractswere prepared 48 h after transfection and dialysed against D buffer. TAT-SF1 complexeswere af®nity puri®ed from the dialysed extracts by incubating with anti-Flag antibodybeads (Sigma), followed by extensive washes with D buffer and Flag peptide elution.

Af®nity puri®cation of U2 snRNP

U2 snRNP was af®nity puri®ed essentially as described25. Brie¯y, the Q fraction in D bufferwas incubated with 2 nmol ml-1 biotinylated 29O-methyl-RNA antisense oligonucleotide(59-GGCCGAGAAGCGAUdT-biotin-39) with sequences complementary to the ®rst 14nucleotides of U2 snRNA. The reaction mix was incubated at room temperature for30 min and then mixed for 1.5 h at 4 8C with pre-blocked streptavidin agarose beads(Sigma). After washing with D buffer, U2 snRNP was eluted by the addition of a 2.5-foldexcess of a displacement DNA oligonucleotide (59-ATCGCTTCTCGGCC-39) in D buffercontaining 0.1% NP40 and 1 mg ml-1 BSA and then concentrated.

Coupled in vitro transcription and splicing

A 336-bp Ad2 fragment derived from pSPAd28 containing a shortened intron and the ®rstand second leader exons were ampli®ed by PCR and inserted between the XbaI and BamHIsites of the template pHIV+TAR-G40019. Similarly, a 212-bp b-globin exon±intron±exonfragment was subcloned into the XbaI and BamHI sites of pHIV+TAR-G400. Varioussplice-site mutant fragments were generated by PCR using the sets of the followingoligonucleotides: (1) 59-CATGTCTAGACTGCGAGGGCCAGCTG-39; (2) 59-CATGTCTAGACTGCGAGGGCCAGCTGTTGGTCATGTGACTCCCTCTCAAAA-39; (3) 59-CTAGGATCCCGTTCGGAGGCCGACGGG-39; (4) 59-GTCCCTTTTTTTTCCAACTCTCGCGGTTGAGGACAAACT-39; (5) 59-AGTTTGTCCTCAACCGCGAGAGTTGGAAAAAAAAGGGAC-39; (6) 59-GATGATGTCATACTTATCAGGAGGAAAGGAAAAGGACAGCTCGCGGTTGAGGAC-39; (7) 59-GTCCTCAACCGCGAGCTGTCCTTTTCCTTTCCTCCTGATAAGTATGACATCATC-39; (8) 59-GGTTTCCTTCTACTGGTCATACTTTAGCTGTCCCTTTTTTTTCCA-39; (9) 59-TGGAAAAAAAAGGGACAGCTAAAGTATGACCAGTAGAAGGAAACC-39; (10) 59-CTTGTTGTTGTCATACTTTAGAGGAGGAAAGGAAAAGGAACTCTCGCGGTT-39; (11) 59-AAGTATGACAACAACAAGGAAACCCTGGACAACAGCGCCCTAGACGTGC-39; (12) 59-CATGTCTAGAAGTTGGTGGTGAGGCCCTG-39; (13) 59-CATGTCTAGAAGTTGGTGGTGAGGCCCTGGGCTCAGCTACATCAAGGTTACAAGACAGG-39; (14) 59-CTAGGATTCGACAGATCCCCAAAGGACTC-39; (15) 59-TTCTGTTAGGGCACTTTGTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTGTCCTGCTGG-39; (16) 59-ACAAAGTGCCTAACAGAAACCCAA-39. The following fragments were generated: Ad-w, oligonucleotides 1 and 3;Ad-39ss, 1, 5, 3 and 4; Ad-59ss, 2 and 3; Ad-Py-t, 1, 7, 3 and 6; Ad-Bp, 1, 9, 3 and 8; Ad-m, 2,11, 3 and 10; b-globin-w, 12 and 14; and b-globin-m, 13, 16, 14 and 15. The transcriptiontemplates used in the assay were linearized with NotI and BamHI or with NotI and XbaI togenerate runoff transcripts of ,0.8 kb (Ad2), ,0.7 kb (b-globin) and ,0.5 kb (internalcontrol). In vitro transcription condition was modi®ed to allow more-ef®cient splicing.Brie¯y, the reaction was scaled up to 25 ml, containing 2% PEG3300, 3.6 mM MgCl2,

0.3 mM ATP, 50 mM GTP, 50 mM UTP, 6.25 mM CTP, 10 mCi [a-32P]CTP (800 Ci mmol-1;1 ml), 25 ng linearized templates, and 30±50 mg HeLa nuclear extract.

Received 14 August; accepted 17 October 2001.

1. Bentley, D. Coupling RNA polymerase II transcription with pre-mRNA processing. Curr. Opin. Cell

Biol. 11, 347±351 (1999).

2. Hirose, Y. & Manley, J. L. RNA polymerase II and the integration of nuclear events. Genes Dev. 14,

1415±1429 (2000).

3. McCracken, S. et al. The C-terminal domain of RNA polymerase II couples mRNA processing to

transcription. Nature 385, 357±361 (1997).

4. Fong, N. & Bentley, D. L. Capping, splicing, and 39 processing are independently stimulated by RNA

polymerase II: different functions for different segments of the CTD. Genes Dev. 15, 1783±1795

(2001).

5. Hirose, Y., Tacke, R. & Manley, J. L. Phosphorylated RNA polymerase II stimulates pre-mRNA

splicing. Genes Dev. 13, 1234±1239 (1999).

6. Kim, J. B., Yamaguchi, Y., Wada, T., Handa, H. & Sharp, P. A. Tat-SF1 protein associates with RAP30

and human SPT5 proteins. Mol. Cell. Biol. 19, 5960±5968 (1999).

7. Li, X. Y. & Green, M. R. The HIV-1 Tat cellular coactivator Tat-SF1 is a general transcription

elongation factor. Genes Dev. 12, 2992±2996 (1998).

8. Zhou, Q. & Sharp, P. A. Tat-SF1: cofactor for stimulation of transcriptional elongation by HIV-1 Tat.

Science 274, 605±610 (1996).

9. Parada, C. A. & Roeder, R. G. A novel RNA polymerase II-containing complex potentiates Tat-

enhanced HIV-1 transcription. EMBO J. 18, 3688±3701 (1999).

10. Price, D. H. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol.

Cell. Biol. 20, 2629±2634 (2000).

11. Ping, Y. H. & Rana, T. M. Tat-associated kinase (P-TEFb): a component of transcription preinitiation

and elongation complexes. J. Biol. Chem. 274, 7399±7404 (1999).

12. Zhou, M. et al. Tat modi®es the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II

carboxyl-terminal domain during human immunode®ciency virus type 1 transcription. Mol. Cell.

Biol. 20, 5077±5086 (2000).

13. Dahmus, M. E. Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J. Biol.

Chem. 271, 19009±19012 (1996).

14. Wei, P., Garber, M. E., Fang, S. M., Fischer, W. H. & Jones, K. A. A novel CDK9-associated C-type

cyclin interacts directly with HIV-1 Tat and mediates its high-af®nity, loop-speci®c binding to TAR

RNA. Cell 92, 451±462 (1998).

15. Jones, K. A. Taking a new TAK on tat transactivation. Genes Dev. 11, 2593±2599 (1997).

letters to nature

932 NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com© 2001 Macmillan Magazines Ltd

16. Garber, M. E. et al. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a

critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev. 12, 3512±3527

(1998).

17. Peng, J., Zhu, Y., Milton, J. T. & Price, D. H. Identi®cation of multiple cyclin subunits of human

P-TEFb. Genes Dev. 12, 755±762 (1998).

18. Fong, Y. W. & Zhou, Q. Relief of two built-in autoinhibitory mechanisms in P-TEFb is required for

assembly of a multicomponent transcription elongation complex at the human immunode®ciency

virus type 1 promoter. Mol. Cell. Biol. 20, 5897±5907 (2000).

19. Zhou, Q. & Sharp, P. A. Novel mechanism and factor for regulation by HIV-1 Tat. EMBO J. 14, 321±

328 (1995).

20. Marciniak, R. A. & Sharp, P. A. HIV-1 Tat protein promotes formation of more-processive elongation

complexes. EMBO J. 10, 4189±4196 (1991).

21. Yan, D. et al. CUS2, a yeast homolog of human Tat-SF1, rescues function of misfolded U2 through an

unusual RNA recognition motif. Mol. Cell. Biol. 18, 5000±5009 (1998).

22. Perriman, R. & Ares, M. Jr ATP can be dispensable for prespliceosome formation in yeast. Genes Dev.

14, 97±107 (2000).

23. Krainer, A. R. Pre-mRNA splicing by complementation with puri®ed human U1, U2, U4/U6 and U5

snRNPs. Nucleic Acids Res. 16, 9415±9429 (1988).

24. Bach, M., Bringmann, P. & LuÈhrmann, R. Puri®cation of small nuclear ribonucleoprotein particles

with antibodies against modi®ed nucleosides of small nuclear RNAs. Methods Enzymol. 181, 232±257

(1990).

25. Blencowe, B. J. & Lamond, A. I. Puri®cation and depletion of RNP particles by antisense af®nity

chromatography. Methods Mol. Biol. 118, 275±287 (1999).

26. Schnapp, G., Rodi, H. P., Rettig, W. J., Schnapp, A. & Damm, K. One-step af®nity puri®cation

protocol for human telomerase. Nucleic Acids Res. 26, 3311±3313 (1998).

27. Krainer, A. R. & Maniatis, T. Multiple factors including the small nuclear ribonucleoproteins U1 and

U2 are necessary for pre-mRNA splicing in vitro. Cell 42, 725±736 (1985).

28. Solnick, D. Alternative splicing caused by RNA secondary structure. Cell 43, 667±676 (1985).

29. Padgett, R. A., Mount, S. M., Steitz, J. A. & Sharp, P. A. Splicing of messenger RNA precursors is

inhibited by antisera to small nuclear ribonucleoprotein. Cell 35, 101±107 (1983).

30. Ares, M. Jr, Grate, L. & Pauling, M. H. A handful of intron-containing genes produces the lion's share

of yeast mRNA. RNA 9, 1138±1139 (1999).

Acknowledgements

We thank B. Blencowe for the gift of antisense RNA oligonucleotide and advice on thedepletion procedures; E. Labourier, D. Rio, J. Underwood, D. Black and H. Wu forreagents; E. Logue and L. Zhou for technical assistance; and K. Luo, I. von Reis andS. Stroschein for discussions. Supported by grants from the National Institutes of Healthto Q.Z. and the University of California Universitywide AIDS Research Program to Y.W.F.

Correspondence and requests for materials should be addressed to Q.Z.(e-mail: qzhou@uclink4.berkeley.edu).

letters to nature

NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com 933

.................................................................Crystal structure of anEph receptor±ephrin complexJuha-Pekka Himanen*, Kanagalaghatta R. Rajashankar²,Martin Lackmann³, Chad A. Cowan§, Mark Henkemeyer§& Dimitar B. Nikolov*

* Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering

Cancer Center, 1275 York Avenue, New York, New York 10021, USA² Brookhaven National Laboratory, Brookhaven, New York 11973, USA³ Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050,

Australia

§ Center for Developmental Biology and Kent Waldrep Foundation Center for

Basic Research on Nerve Growth and Regeneration, University of TexasSouthwestern Medical Center, Dallas, Texas 75390-9133, USA

..............................................................................................................................................

The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell±cellinteractions as they initiate a unique bidirectional signal trans-duction cascade whereby information is communicated into boththe Eph-expressing and the ephrin-expressing cells. Initiallyidenti®ed as regulators of axon path®nding and neuronal cellmigration, Ephs and ephrins are now known to have roles in manyother cell±cell interactions, including those of vascular endothe-lial cells and specialized epithelia1,2. Here we report the crystalstructure of the complex formed between EphB2 and ephrin-B2,determined at 2.7 AÊ resolution. Each Eph receptor binds an ephrin

ligand through an expansive dimerization interface dominated bythe insertion of an extended ephrin loop into a channel at thesurface of the receptor. Two Eph±Ephrin dimers then join to forma tetramer, in which each ligand interacts with two receptors andeach receptor interacts with two ligands. The Eph and ephrinmolecules are precisely positioned and orientated in these com-plexes, promoting higher-order clustering and the initiation ofbidirectional signalling.

The communication of biochemical signals between cells isessential for the development and existence of multicellular organ-isms. A key method of communication is based on direct protein±protein interactions between ligands, which carry the signal, andcell-surface receptors, which recognize and transduce the informa-tion into the receiving cell. One large group of receptors and ligands,the Eph/ephrin family, sends information bidirectionally into boththe receptor-expressing cell and the ligand-expressing cell3±6. Ephsand ephrins are divided into two subclasses, A and B, according totheir af®nities for each other, sequence conservation, and the modeof ephrin membrane attachment (refs 7, 8; and Fig. 1). In general,the EphA receptors (EphA1 to EphA8) promiscuously bind to andare activated by glycosyl phosphatidylinositol (GPI)-anchored A-ephrins (ephrin-A1 to ephrin-A5), whereas EphB receptors (EphB1to EphB6, and in some cases EphA4) bind to and are activated by thetransmembrane B-ephrins (ephrin-B1 to ephrin-B3).

The membrane attachment of both Ephs and ephrins provides amechanism whereby interactions between receptors and ligandsoccur only at sites of cell±cell contact, leading to the multimeriza-tion of both molecules to distinct clusters in their respective plasmamembranes9,10. This results in the formation of discrete bidirec-tional signalling centres in which the Eph receptor tyrosine kinasedomain transduces the forward signal into its cell and the ephrintransduces the reverse signal into its cell. Although crystal structuresfor the isolated EphB2 and ephrin-B2 extracellular domains havebeen described11,12, the precise molecular determinants that governsubclass speci®city, higher-order clustering, and bidirectionalsignalling have not been identi®ed. We therefore generated co-crystals and determined the structure of ephrin-B2 bound toEphB2.

The amino-terminal ligand-binding globular domain of EphB2(refs 11, 13, 14) and the complete extracellular domain of ephrin-B2were expressed in Escherichia coli and crystallized (see Methods).The structure was determined using multiwavelength anomalousdiffraction15 (see Supplementary Information), and is re®ned at2.7 AÊ resolution.

Biophysical experiments indicate that the extracellular domainsof Eph receptors and ephrins interact by a multiple-step process(refs 14, 16 and 17, and J.P.H. and D.B.N., manuscript in prepara-tion). Ephs and ephrins ®rst bind with high af®nity and speci®cityto form heterodimers, which are the predominant form of thecomplex in solution throughout a large concentration range from10 nM to 20 mM. At higher concentrations, the dimer pairs associateinto tetramers and higher-order aggregates.

The dimeric and tetrameric structures are both present in ourcrystals. The tetrameric complex forms a ring-like structure, inwhich each ligand interacts with two receptors and each receptorwith two ligands (Fig. 2a, b). One of the ligand±receptor interfacesis very extensive and presumably mediates the initial high-af®nity1:1 dimerization measured in solution (arrows in Fig. 2a). Thesecond interface is smaller (Fig. 2a) and may be responsible for theassembly of functional tetrameric ligand±receptor complexes. Thecarboxy termini of both ephrin-B2 molecules are located at one sideof the tetramer, whereas the carboxyl termini of both EphB2molecules are located at the other side (Fig. 2b). Such a structureis consistent with the expected positioning and orientation of boththe ligand and receptor molecules on the surfaces of adjacent,juxtaposed cells.

The overall structure of ephrin-B2 in the complex (Fig. 3a, b) is

© 2001 Macmillan Magazines Ltd