Spliceosome Dynamics I.pdf

Subunit remodelingPrp3Prp4PPIHLSmSmSnu13Prp31U4U6Brr2U2E1pre-mRNAU6Protein exchange(human)A complexPROTEIN LISTU2U1U1U5U4U6U2B complex Bact complexU2U5U6U2U5U6C complexReactive sitesExon 1Exon 2GURAGUYNYURAYY10-12YAGUACUAACAGGUAAGU 48 nt 18 nt5SS3SSBPSPolypyrimidinetractMetazoaYeastSplicing chemistry-35-5SS3SSBPApE1E2p-35-AOHE1E2ppStep 1Step 2-35-E1E2pApOH+mRNAExcised intron-lariatRNA rearrangementsPre-catalytic spliceosomeCatalytically activatedspliceosomeU2AACAGAACAGAU1U4U6E1E2U5U2U6-ISLU6E2E1AU5Catalytic RNA network on Prp8ACAGAGAAGAG53U2pre-mRNAU6U53535E1U6U2U5pre-mRNAPrp8A complex(pre-spliceosome)U2U1 Bact complex(activatedspliceosome)U2U5U6U5U6U2C complex(catalytic step 1spliceosome)Step 1U1U5U4U6U2B complex(pre-catalyticspliceosome)Brr2Snu114Step 2Prp16Prp28Prp5Sub2/UAP56 B*complex(catalyticallyactivatedspliceosome)U2U5Prp2IBCRESEJCU1SF1E complexB complexproteinsCross-intronassemblyPrp19NPCNUCLEUSCYTOPLASMU2U6U5Post-splicingcomplexmRNPPrp22Intron-lariatcomplexU2U5U6Intron-lariatU5U6Prp43Crosstalk tochromatinCo-transcriptionalassemblypre-mRNAkonkoffPol IICTDChromatinSplicingregulatoryfactorsU5U4U6U4U6U1U4U5U2U1SART3/Prp24Compositions of major subunits(human)Prp19Prp19CDC5SPF27PRL1AD-002Catenin,-like 1Hsp73IBCAquarius hSyf1 CCDC16hIsy1 CypERESMGC13125SNIPCGI-79AcinuseIF4A3Y14MagohAlyUAP56THOC1-3EJCU1 snRNA70KU1ASmcoreU1 snRNPSm/LSmSmSmSmhSnu13hPrp31hPrp3hPrp4PPIHU6 U4U6U4U6 U4U5Sm/LSmhPrp8hBrr2hSnu11440KhPrp6hDib1hPrp2827KhSad1hSnu66hSnu13hPrp31hPrp3hPrp4PPIHU5U6U470KACU1U1ABSF3a120SF3a66SF3a60SF3b155U2U2hPrp8hBrr2hSnu11440KhPrp6hLin1hDib1hPrp28U5U5Small nuclear ribonucleoprotein particles(snRNPs)Multi-protein subunitsContext-dependentrecruitmentSF3b145SF3b130SF3b49SF3b14aSF3b14bSF3b10U6U2U1A complexproteinsBUB3MGC2803SF4FLJ10839CDC2L2Tat SF1TLS/FUSRMB5RBM10SF1U2U1 snRNASm70KACU2 snRNASmA,BSF3a(3)SF3b(7)U2 relatedU2AF65+35PUF60SPF30SPF31SPF45CHERPSR140hPrp43hPrp5hnRNPproteinsSR proteinsmRNP proteinsFBP11S164p68CA150Prp19complexU4/U6U5tri-snRNPREScomplexMissing hPrp5 KIAA1604,PPIL2,CCDC12 onlyMGC13125SNIPCGI-79Prp19CDC5SPF27PRL1AD-002Catenin,-like 1Hsp73Prp19relatedSKIPhSyf3PPIL1RBM22G10U5U4/U6U5 snRNASmhSnu114hBrr2hPrp840KhPrp6hPrp28hDib1U6 snRNALSm 2-8U4 snRNASmhPrp31PPIHhPrp4hPrp3hSnu1327KhSad1hSnu66AquariushSyf1CCDC16hIsy1CypEIBCNPW38NPW38BPHSP27Hskin17Skiv2L2hPRP4 kinhSnu23hPrp38MFAP1UBL5REDhSmu1KIAA1604PPIL2CCDC12RNF113AhPrp17NY-CO-10PPIL3bhPrp2hSPP2Bact complexproteinsB complexproteinsStep 2factorshPrp17Slu7hPrp22hPrp18hPrp16C complexproteinsAbstraktCactinDDX35O9BRR8PPWD1GCIP p29CXorf56C9orf78PPIGFRA10AC1FRG1MORG1DGCR14NOSIPC1orf55FAM50A+BFAM32ASrm300*U6 snRNA onlyeIF4A3,Y14,Magoh only*U6 snRNA only*AcinuseIF4A3Y14MagohAlyUAP56THOC13EJC/TREX*SnapShot:Spliceosome Dynamics IMarkus C.Wahl1 and Reinhard Lhrmann21Laboratory of Structural Biochemistry,Freie Universitt Berlin,Takustrae 6,14195 Berlin,Germany2Department of Cellular Biochemistry,Max Planck Institute for Biophysical Chemistry,Am Fassberg 11,37077 Gttingen,GermanySee online version for legend and references.1474 Cell 161,June 4,2015 2015 Elsevier Inc.DOI http:/dx.doi.org/10.1016/j.cell.2015.05.0501474.e1 Cell 161,June 4,2015 2015 Elsevier Inc.DOI http:/dx.doi.org/10.1016/j.cell.2015.05.050SnapShot:Spliceosome Dynamics IMarkus C.Wahl1 and Reinhard Lhrmann21Laboratory of Structural Biochemistry,Freie Universitt Berlin,Takustrae 6,14195 Berlin,Germany2Department of Cellular Biochemistry,Max Planck Institute for Biophysical Chemistry,Am Fassberg 11,37077 Gttingen,GermanySpliceosomes are multi-megadalton RNA-protein molecular machines that carry out pre-mRNA splicing,that is,the removal of non-coding intervening sequences(introns)from eukaryotic pre-mRNAs and the ligation of neighboring coding regions(exons)to produce mature mRNA for protein biosynthesis on the ribosome.They are the prototypes of dynamic molecular machines,assembling de novo for each splicing event by the stepwise recruitment of subunits on a substrate.Intron excision and exon ligation by spliceosomes are achieved via two consecutive transesterification reactions involving the 5-splice site(SS),a branch point sequence(BPS),and the 3SS(Wahl et al.,2009).In vivo,spliceosomes typically assemble on a substrate while it is still being transcribed by RNA polymerase II(Pol II),and early splicing factors are recruited to the pre-mRNA through the Pol II C-terminal domain(CTD)(Kornblihtt et al.,2013).Pol II and the spliceosome mutually influence each other;for example,in yeast,transcriptional pausing at the 3 ends of introns coincides with splicing factor recruitment/splicing and,vice versa,Pol II pausing depends on splicing.Co-transcriptional splicing is also influenced by chromatin organization,and splicing can modulate histone marks(de Almeida et al.,2011;Luco et al.,2011).In addition,splicing is physically and functionally coupled to other pre-mRNA processing events,such as 5-capping and 3-cleavage/polyadenylation(Maniatis and Reed,2002).Furthermore,there is crosstalk of the spliceosome to other mRNA metabolic processes,for example,mRNA export and cytoplasmic surveillance/translation via recruitment of export factors and the exon junction complex(EJC)during splicing(Wahl et al.,2009).The main spliceosome assembly process entails the stepwise recruitment of RNA-protein subunits,small nuclear(sn)RNPs U1,U2,U4,U5,and U6 in case of the major spliceosome,and a plethora of non-snRNP proteins(central pathway).Substrate-specific splicing regulators may be integrated in response to cis-acting elements on the pre-mRNA(Wahl et al.,2009),likely with context-dependent integration kinetics.The snRNPs each contain a specific snRNA,a common set of seven Sm(in U1,U2,U4,and U5 snRNPs)or LSm(in U6 snRNP)proteins and a variable number of particle-specific proteins(Wahl et al.,2009).Some non-snRNP proteins are also pre-organized as multimeric complexes,for example,the Prp19 complex(Prp19),the intron-binding complex(IBC)or the pre-mRNA retention and splicing complex(RES).During canonical cross-intron assembly,U1 snRNP recognizes the 5SS(complex E)and U2 snRNP replaces early-binding SF1 protein at the BPS,forming complex A.Subsequently,the U4,U5,and U6 snRNPs join as a pre-formed tri-snRNP,giving rise to complex B,which is still inactive.Complex B undergoes major compositional and conformational remodeling in several steps(forming complexes Bact and subsequently B*)to become catalytically competent.The B*complex can then carry out the first step of splicing;the ensuing C complex facilitates the second step before the spliceosome is disassembled in an ordered fashion.Thus,the spliceosomes active site is a fleeting entity that only emerges transiently during assembly on a substrate.It is thought that splicing catalysis is largely an RNA-based process facilitated by the emerging U2-U5-U6-pre-mRNA network(Anokhina et al.,2013;Fica et al.,2013),but proteins,such as the large Prp8 platform(Galej et al.,2013),are essential for the formation of the active site.At all transitions in the splicing process,the spliceosomes underlying RNA-protein interaction network is compositionally and conformationally remodeled and at each step there is a massive exchange of proteins(Wahl et al.,2009).These remodeling events are mediated by conserved spliceosomal RNA helicases and a G protein,Snu114,which resembles the ribosomal translocase,EF-G/eEF2(blue along the pathway)(Staley and Guthrie,1998).In addition,post-translational modification enzymes and peptidyl-prolyl cis/trans isomerases are associated with the spliceosome and might implement additional remodeling steps;for example,human SF3b155 and CDC5L proteins are phosphory-lated precisely during the B-to-Bact and Bact-to-C transition,respectively.The multitude of putative remodeling enzymes associated with spliceosomes suggests that many more assembly intermediates/steps exist;for example,for many groups of non-snRNP proteins,which join during the same phase,it is poorly understood to what extent recruitment is coordinated or sequential.Higher eukaryotes contain additional helicases and putative remodeling enzymes(De et al.,2015),suggesting that they in general assemble via a larger number of steps and offer more possibilities for regulation.Other kinds of conformational switches seem to depend on proteins without enzymatic remodeling activity;for example,B complex-specific proteins enter the spliceosome only during the B complex stage and may serve as transient scaffolds that facilitate productive organization of other components.Remodeling of the spliceosome during splicing extends all the way to the level of the snRNPs;for example,U4/U6 di-snRNP is completely disrupted during catalytic activation(Wahl et al.,2009).As a consequence,several snRNPs have to be re-assembled after each splicing reaction to be able to engage in further rounds of splicing.Subunit recycling requires assembly chaperones such as the SART3 protein in human or the Prp24 protein in yeast for U4/U6 re-assembly(Staley and Guthrie,1998).RefeRencesAnokhina,M.,Bessonov,S.,Miao,Z.,Westhof,E.,Hartmuth,K.,and Lhrmann,R.(2013).EMBO J.32,28042818.De,I.,Bessonov,S.,Hofele,R.,dos Santos,K.,Will,C.L.,Urlaub,H.,Lhrmann,R.,and Pena,V.(2015).Nat.Struct.Mol.Biol.22,138144.de Almeida,S.F.,Grosso,A.R.,Koch,F.,Fenouil,R.,Carvalho,S.,Andrade,J.,Levezinho,H.,Gut,M.,Eick,D.,Gut,I.,et al.(2011).Nat.Struct.Mol.Biol.18,977983.Fica,S.M.,Tuttle,N.,Novak,T.,Li,N.S.,Lu,J.,Koodathingal,P.,Dai,Q.,Staley,J.P.,and Piccirilli,J.A.(2013).Nature 503,229234.Galej,W.P.,Oubridge,C.,Newman,A.J.,and Nagai,K.(2013).Nature 493,638643.Kornblihtt,A.R.,Schor,I.E.,All,M.,Dujardin,G.,Petrillo,E.,and Muoz,M.J.(2013).Nat.Rev.Mol.Cell Biol.14,153165.Luco,R.F.,Allo,M.,Schor,I.E.,Kornblihtt,A.R.,and Misteli,T.(2011).Cell 144,1626.Maniatis,T.,and Reed,R.(2002).Nature 416,499506.Staley,J.P.,and Guthrie,C.(1998).Cell 92,315326.Wahl,M.C.,Will,C.L.,and Lhrmann,R.(2009).Cell 136,701718.