1、Molecular Cell, Vol. 19, 5363, July 1, 2005, Copyright 2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.05.007cently, this question has been difficult to address. Re-base-pairing interactions (Staley and Guthrie, 1998).sults from metazoan systems indicate that splicing atWhether these dynamics are ca
2、rried out within a pre-least begins cotranscriptionally (Neugebauer, 2002),formed spliceosomal machine or whether they reflectraising the possibility that spliceosome assembly canthe addition and loss of individual spliceosomal com-be monitored in vivo by examining the association ofponents bound to
3、 the pre-mRNA is an unresolvedsplicing factors along the lengths of genes. Becausequestion.nascent RNPs lie adjacent to the DNA axis (Wetterberget al., 2001), the use of formaldehyde to efficientlyinduce protein-protein and protein-nucleic acid cross-*Correspondence: neugebaumpi-cbg.deCotranscriptio
4、nal SpliceosomOccurs in a Stepwise Fashionthe Cap Binding ComplexJanina Grnemann, Kimberly M. Kotovic,Katja Hujer, and Karla M. Neugebauer*Max Planck Institute of Molecular Cell Biologyand GeneticsPfotenhauerstrasse 10801307 DresdenGermanySummaryCoupling between transcription and pre-mRNA splic-ing
5、is a key regulatory mechanism in gene expres-sion. Here, we investigate cotranscriptional spliceo-some assembly in yeast, using in vivo crosslinking todetermine the distribution of spliceosome compo-nents along intron-containing genes. Accumulationof the U1, U2, and U5 small nuclear ribonucleoprotei
6、nparticles (snRNPs) and the 3H11541 splice site binding fac-tors Mud2p and BBP was detected in patterns indica-tive of progressive and complete spliceosome assem-bly; recruitment of the nineteen complex (NTC)component Prp19p suggests that splicing catalysis isalso cotranscriptional. The separate dyn
7、amics of theU1, U2, and U5 snRNPs are consistent with stepwiserecruitment of individual snRNPs rather than a pre-formed “penta-snRNP,” as recently proposed. Finally,we show that the cap binding complex (CBC) is nec-essary, but not sufficient, for cotranscriptional spli-ceosome assembly. Thus, the de
8、monstration of anessential link between CBC and spliceosome assem-bly in vivo indicates that 5H11541 end capping couples pre-mRNA splicing to transcription.IntroductionPre-mRNA splicing is carried out by the spliceosome,a multicomponent complex approximately the size ofthe ribosome (Jurica and Moore
9、, 2003; Nilsen, 2003).The two-step trans-esterification reaction, which leadsto cleavage of the RNA at intron-exon boundaries andthe ligation together of two adjacent exons, dependsupon the sequential activities of the spliceosomal smallnuclear RNAs (snRNAs) U2, U4, U5, and U6. Prior tosplicing, rec
10、ognition of the 5# and 3# splice sites by theU1 and U2 snRNAs, augmented by snRNP and non-snRNP splicing factors, serves to define the splice sitesand to stimulate spliceosome assembly. Transitionsfrom splice site recognition to the initiation of splicingand during catalysis are marked by profound r
11、e-arrangements in snRNA-pre-mRNA and snRNA-snRNAe Assemblyand RequiresUntil recently, it has been assumed that spliceosomeassembly occurs in a step-wise fashion, with the U1snRNP and the U2 snRNP added independently topre-mRNA, followed by the addition of the U4/U6 U5tri-snRNP (Reed, 2000). The U1,
12、U2, and U4/U6 U5 tri-snRNPs can be isolated individually from nuclear ex-tracts, which are capable of splicing pre-mRNA in thepresence of ATP. Moreover, distinct intermediates inspliceosome assembly can be detected and purified. Inyeast and mammals, early ATP-independent assemblysteps include the fo
13、rmation of commitment or E com-plexes, reflecting pre-mRNA association with the U1snRNP and the 3# splice site factors BBP (SF1 in mam-mals) and Mud2p (U2AF65 in mammals) (Abovich andRosbash, 1997; Michaud and Reed, 1991; Seraphin andRosbash, 1989). After the addition of ATP to in vitrosplicing reac
14、tions, the U2 snRNP joins to form mamma-lian complex A, and further separable steps in spliceo-some assembly can be arrested (Chiara et al., 1996).Purification of these complexes in biochemical amountshas permitted their proteomic characterization by massspectrometry, providing a detailed list of pr
15、oteins thatparticipate in the splicing reaction (Jurica et al., 2002;Makarov et al., 2002; Rappsilber et al., 2002; Zhou etal., 2002).However, it has recently been shown in yeast that,depending on the salt concentration used in preparingthe nuclear extract, a larger 45S particle containing allof the
16、 spliceosomal snRNPs is present (Stevens et al.,2002). This has given rise to the penta-snRNP hypothe-sis, which holds that all five spliceosomal snRNPs arerecruited to pre-mRNA together; within this super com-plex of pre-mRNA and spliceosomal components, all ofthe aforementioned dynamics between pr
17、e-mRNA andsnRNAs may take place. Support for the penta-snRNPhypothesis has come from in vitro evidence in highereukaryotes that the U1 and U5 snRNPs contact the 5#splice site independently of 3# splice site recognitionand/or in the context of a mammalian penta-snRNP(Malca et al., 2003; Maroney et al
18、., 2000; Wyatt et al.,1992). Moreover, it has long been known that large200S complexes, containing all of the spliceosomalsnRNPs, most non-snRNP splicing factors, and pre-mRNA, can be isolated by gradient centrifugation ofHeLa nuclear extracts (Miriami et al., 1995; Raitskin etal., 2002). This mamma
19、lian complex, now termed thesupraspliceosome, may accommodate multiple penta-snRNPs (Azubel et al., 2004). This body of data hascalled into question the composition of the spliceoso-mal machine and its parts (Nilsen, 2002; Nilsen, 2003).How do spliceosomes assemble in vivo? Until re-Molecular Cell54
20、links among nearby (w2 ) reactive groups (Orlando, 1components2000) can potentially capture spliceosome assemblytrevents that occur cotranscriptionally. Indeed, we foundthein previous work that cotranscriptional association ofORFthe U1 snRNP with intron-containing genes in Sacchar-genesomyces cerevi
21、siae was detectable by chromatin immu-fernoprecipitation (ChIP), in which cells are subjected to1formaldehyde crosslinking prior to cell lysis, DNAintrshearing, immunoprecipitation, and PCR detection ofeventspurified DNA fragments (Kotovic et al., 2003). Thus, themulationChIP approach offers the pos
22、sibility of monitoring fur-shortther events in spliceosome assembly with spatial and,ondby inference, temporal resolution, as nascent RNA isdownstrsynthesized by RNA polymerase II (pol II) from the be-ginning to the end of the gene.rHere, we show by epitope-tagging endogenous genesmidineencoding com
23、ponents of the U2 and U5 snRNPs, thetical,NTC involved in spliceosome activation, and the 3#coincidedsplice site factors Mud2p and BBP (branchpoint bind-uring protein) that spliceosome assembly proceeds toonapparent completion by the time RNA pol II hasbranchpointreached the end of intron-containing
24、 genes. Moreover,ofthe distinct patterns of accumulation observed indicatesequencesthat individual components of the spliceosome are re-withcruited in a step-wise fashion. Because 5# end cappingwerof mRNAs also occurs cotranscriptionally (Shuman,components2001), and because the nuclear CBC binds the
25、 cap co-detectiontranscriptionally and is thought to stimulate splicing(Dower and Rosbash, 2002; Izaurralde et al., 1994; LeMud2p,Hir et al., 2003; Visa et al., 1996; Zenklusen et al., 2002),levelscotranscriptional spliceosome assembly was examinedintrin a strain bearing viable deletions of both sub
26、unitswof CBC (CBP20 and CBP80). Two opposing models foraCBC function in splicing have been proposed: (1) thatMud2p,CBC may promote early steps in splicing, such as com-turmitment complex formation and/or U1 snRNP recruit-bothment (Abovich et al., 1994; Colot et al., 1996; Fortes etofal., 1999a; Fort
27、es et al., 1999b; Lewis et al., 1996a;exonLewis et al., 1996b), or (2) that CBC may facilitate re-ECM33moval the U1 snRNP by the helicase Prp28 upon asso-U1ciation of the tri-snRNP (Chen et al., 2001; OMullanetionallyand Eperon, 1998). We provide evidence for both ofcorrthese roles of CBC, which are
28、 differentially detected onrthe genes under study. On every gene examined here,U5 snRNP accumulation was nearly abolished, indicat-allying that CBC is required for proper coupling of splicingsnRNPto transcription in yeast.Tsults,tected.Resultsteins1As a first step toward examining cotranscriptional
29、spli-Onceosome assembly, we asked whether the commitmentpeak,complex components Mud2p, BBP, and the U2 snRNPsitewere detectable on intron-containing genes by ChIP.andStrains were created in which the endogenous genesintrencoding Mud2p, BBP, and the U2 snRNP-specific pro-earlierteins Lea1p and Msl1p
30、were epitope tagged in angionsotherwise wild-type (wt) background. ChIP was per-ECM33formed with tag-specific monoclonal antibodies (mAbs)and with 8WG16 specific for RNA pol II as a positivecontrol and internal reference; immunopurified DNAwas detected and quantified by real-time PCR. Figureshows th
31、at all of the tagged commitment complexwere detectable on two endogenous in-on-containing genes, ECM33 and DBP2-GFP, in whichDBP2 gene was lengthened by tagging the DBP2with the green fluorescent protein (730 bp). Thesediffer in their structure and therefore provide dif-ent sets of information (see
32、gene diagrams in Figure). DBP2 has a very long first exon (1273 bp) and longon (1003 bp), facilitating the separation of earlyin splicing factor recruitment from pol II accu-at the promoter. In contrast, ECM33 has afirst exon and short intron, whereas the long sec-exon (1348 bp) provides additional
33、resolution ineam regions.The distributions of Mud2p and BBP, which bind di-ectly to 3# splice site intronic elements (the polypyri-tract and branchpoint, respectively) were iden-reaching maximum signals over gene regions thatwith the accumulation of the U1 snRNP (Fig-e 1, top four panels). Interesti
34、ngly, the observed peakDBP2 precedes somewhat the position of thesequence, suggesting that either or boththese proteins may interact with additional intronicin this pre-mRNA. Note that ChIP signalstagged Prp42p, one component of the U1 snRNP,e previously shown to coincide with other taggedof the U1
35、snRNP, establishing that Prp42pis indicative of intact U1 snRNP accumulationKotovic et al., 2003). In DBP2-GFP, the U1 snRNP,and BBP were enriched 5-fold over promoterat a position corresponding to the middle of theon, whereas each of these factors was enriched20-fold at the ECM33 3# splice site as
36、compared toregion 500 bp upstream of the promoter. U1 snRNP,and BBP declined in downstream regions, re-ning to very low levels in downstream regions ofDBP2-GFP and ECM33. The distribution of eachthese factors on SAC6, a gene with similar intron/structure to ECM33, was similar to that shown for(data
37、not shown). We conclude from this thatsnRNP, Mud2p, and BBP accumulate cotranscrip-in identical patterns, suggesting that theirelated appearance on intron-containing genes rep-esents commitment complex formation.The U2 snRNP was also detectable cotranscription-but accumulated in a pattern distinct f
38、rom U1, Mud2p, and BBP (Figure 1, lower two panels).agged Lea1p and Msl1p proteins yielded identical re-indicating that the intact U2 snRNP was de-On DBP2-GFP, the ChIP signals for both pro-peaked 200 bp downstream of the 3# splice site,kb after the peak of the U1 snRNP, Mud2p, and BBP.ECM33, the U2
39、 snRNP was detected in a broadbeginning 100 bp downstream of the 3# spliceand initially overlapping with the U1 snRNP, Mud2p,BBP; this overlap is expected, because the ECM33on is only 331 bp long. However, in contrast to thesefactors, U2 snRNP levels remained high in re-downstream of their initial p
40、eak on DBP2-GFP, and SAC6 (data not shown). This indicates thatthe decrease in U1 snRNP, Mud2p, and BBP detectionis not a general phenomenon of decreased accessibilityin downstream gene regions. Taken together, thesedata indicate that U2 snRNP concentrates on intron-Cotranscriptional Assembly of the
41、 Yeast Spliceosome55Figure 1. Cotranscriptional Commitment Complex Formation and U2 snRNP AdditionDiagrams representing the genes DBP2-GFP (A) and ECM33 (B) show the positions of the PCR products used for analysis. In the panelsaligned immediately below these diagrams, histogram bars are placed acco
42、rding to the positions of the PCR products along each gene. Errorbars represent the SD.(A) Whereas the amount of RNA pol II remains constant along DBP2-GFP, the dynamics of splicing factor association can be observed.Components of the commitment complex (PrP42p in the U1 snRNP, BBP, and Mud2p) accum
43、ulate after synthesis of the 5# splice site, andU2 snRNP factors (Lea1p and Ms11p) can be detected after synthesis of the 3# splice site. The levels of accumulation of the analyzed factorsare expressed relative to the promoter-proximal position.(B) Compared to an upstream position (500 bp), RNA poly
44、merase II (pol II) levels increase 20-fold and remain constant along ECM33.Incontrast, splicing factors increase compared to promoter levels after synthesis of the splice sites as in (A).The data represent the average of at least three independent experiments. (A) Prp42p, n = 3; BBP, n = 4; Mud2p, n
45、 = 6; Lea1p, n = 3; andMsl1p,n=3.(B)Prp42p,n=3;BBP,n=4;Mud2p,n=5;Lea1p,n=3;Msl1p,n=3.containing genes after 3# splice site synthesis and per- proteins for tagging. Instead, we tagged Prp19p, oneof w11 proteins comprising the NTC that associatessists after detectability of U1 snRNP, Mud2p, and BBPhas
46、 been lost. Thus, U1 and U2 snRNP dynamics are with the spliceosome after tri-snRNP addition and isthought to activate the spliceosome for catalysis (Chandistinct with respect to each other and with respect totheir position along intron-containing genes. et al., 2003; Makarova et al., 2004; Ohi et a
47、l., 2005; Tarnet al., 1994). Figure 2 shows that all four of these taggedTo extend these results to later steps in spliceosomeassembly, we tagged endogenous genes encoding proteins accumulate cotranscriptionally. On DBP2-GFP,all three U5 snRNP components and Prp19p peaked atthree components of the U
48、5 snRNP (Prp8p, Brr2p, andSnu114p). In this context, we assume the U5 snRNP the most downstream position examined, w500 bp af-marks the arrival of the U4/U6 U5 tri-snRNP and poten-tially remains until catalysis; due to the fact that theU4/U6 snRNP disassembles upon tri-snRNP addition tothe spliceoso
49、me, we did not choose any U4/U6 snRNPter the 3# splice site, reaching levels w10-fold (Brr2pand Snu114p), w8-fold (Prp8p), and w15-fold (Prp19p)above promoter levels. On ECM33, all four proteinsreached their peak signals (w20-fold over upstream)Molecular Cell56Figure 2. Cotranscriptional Formation o
50、f the Active SpliceosomeLevels of U5 snRNP components (Brr2p, Snu114p, and Prp8p) and NTC component Prp19p increase strongly downstream of the 3# splicesite on DBP2-GFP (A) and ECM33 (B). The setup of the figure is the same as in Figure 1. Values were normalized by setting the promoter-proximal posi
