Spliceosome Dynamics II.pdf

U11Intrinsically unstructured elementsExon 1SRU2AF3SSExon 2U1Intron5SSRepeated recognitionExon 1Exon 2GURAGUYNYURAYU2AF65U2AF35RS CRRM2SF1/BBPU2 snRNAU2 snRNPSF3b155p14Exon 1Exon 2GURAGURSCARRM3RRM2+YNYUR YE complexA complexAlternative splicing patternsExon skipping(cassette exon)Alternative 5SSAlternative 3SSMutually exclusiveexonsIntron retentionConstitutivesplicingConstitutiveexonAlternativeexonIntron53Minor(U12 type)spliceosomeSm/Sm20K25K31K35K48K59K65KSF3b155SF3b145SF3b130SF3b49SF3b14aSF3b14bSF3b10U11U12SmhPrp8hBrr2hSnu11440KhPrp6hLin1hDib1hPrp28U5Sm/LSmhSnu13hPrp31hPrp3hPrp4PPIHU6atacU4atacSm/LSmhPrp8hBrr2hSnu11440KhPrp6hDib1hPrp2827KhSad1hSnu66hSnu13hPrp31hPrp3hPrp4PPIHU5U6 U4U6 U4U5U6atacU4atacExon 1Exon 2GURAGUYNYURAYY10-12YAGUAUCCUUU5SS3SSBPSatacatac atacU6 U4atac atacatacU11U12PolypyrimidinetractMajorMinorAGUCCUUAACUYACGA complex(pre-spliceosome)Bact complex(activatedspliceosome)AUACApre-mRNAmRNPIntron-lariatU11U11U11U11U12U12U12U5U6 U4U5U6 U4U5U5U5U4atacU6atacU6atacU6atacU6atacU6atacU4atac/U6atacU5tri-snRNPU5Post-splicingcomplexB complex(pre-catalyticspliceosome)C complex(catalytic step 1spliceosome)5SSBPSStep 1Step 23SS B*complex(catalytically activatedspliceosome)U12U12U12Alternative assembly pathwaysCross-intronA complexU1U1U2Cross-intronB complexOrU1U1U2GUGUCross-exon assembly.SR proteins and hnRNPs35U2AF65U1.hnRNPU2.U1U1U1U2.35U2AF65RSRRMRRMPPPPPPPPPPOrOrSRPKsATPRRMRRMRSRSSR proteinPrp8(Jab1)Brr2IntermediateinhibitionFidelity and proofreadingINITIAL ASSEMBLYACTIVATIONPrp5(optimalsubstrate)Brr2,Snu114(G protein)Prp28(optimalsubstrate)CATALYSISDISASSEMBLYStep 1Prp16(optimalsubstrate)Step 2Prp22(optimalsubstrate)U15SS3SSBPDiscard by?Prp5(sub-optimalsubstrate)E complexU2U1Discard by?Prp28(sub-optimalsubstrate)A complexU2U6B complexPrp2(optimalsubstrate)Discard by?Prp2(sub-optimalsubstrate)SF3bBact complexSF3bB*complexDiscard byPrp43Prp16(sub-optimalsubstrate)C complexDiscard byPrp43Prp22(sub-optimalsubstrate)Post-splicing complexPrp43Intron-lariat complexLong-rangecontacts?SRU15SSLong-rangeY10-12YAGY10-12YAGU5U12U12U6atacU12U5U5U5U6 U4atac atacU5U12U2A YRYYRYYRYYRYYRYYRYAG35U2AF65ESESRGUAGESEGUAAISEISSAGGUESSGUU5U6 U4RRM1RSNRRMRRM1RSRRM3NRRMU2AUGAUGRRMRRMRRMRRMSee online version for legend and references.456 Cell 162,July 16,2015 2015 Elsevier Inc.DOI http:/dx.doi.org/10.1016/j.cell.2015.06.061SnapShot:Spliceosome Dynamics IIMarkus 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,Germany456.e1 Cell 162,July 16,2015 2015 Elsevier Inc.DOI http:/dx.doi.org/10.1016/j.cell.2015.06.061SnapShot:Spliceosome Dynamics IIMarkus 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,GermanyNumerous mechanisms exploit or modulate the conformational/compositional dynamics of spliceosomes to regulate splicing.The majority of higher eukaryotic protein-coding genes contain more than one intron and the derived pre-mRNAs can be alternatively spliced.Diverse principles ensure the reliable identification of authentic splice sites while concomitantly providing flexibility in splice site choice during alternative splicing.Some species contain a second type of minor(U12-type)spliceosome.Various forms of alternative splicing and their combination with alternative transcription initiation sites and/or alternative 3-cleavage/polyadenylation sites vastly increases the number of mature transcripts that can derive from a single protein-coding gene.Kinetic and/or physical coupling of the spliceosome with signal transduction,chromatin organization,and transcription are important mechanisms that regulate alternative splicing(Kornblihtt et al.,2013).During splicing,stepwise assembly is one principle by which spliceosomes on the one hand ensure the faithful recognition of splice sites and on the other hand become susceptible to regulatory inputs to implement alternative splicing(Wahl et al.,2009).During assembly of a spliceosome,all reactive sites on a pre-mRNA are recognized several times by different factors,thus increasing fidelity.Assembly is largely controlled by spliceosome-associated RNA helicases and a G protein(Snu114)that act as ribonucleoprotein(RNP)remodeling enzymes.Similar to kinetic proofreading on the ribosome,the“ideality”of an assembly intermediate may determine how fast a remodeling enzyme can act and drive the intermediate along the productive splicing pathway(Query and Konarska,2006).Thus,mutually exclusive,alternative splicing scenarios may be“serviced”with different kinetics by the spliceosome.Several spliceosomal heli-cases can alternatively channel sub-optimal,slow converting intermediates into discard pathways.In addition to ultimately disassembling the spliceosome,the Prp43 helicase and its NTR1/2 cofactors may act as general discard factors in these proofreading/fidelity mechanisms(Semlow and Staley,2012).By competing with productive splicing,dis-card mechanisms may also influence the balance of alternatively spliced products.The highly variable length and,in some instances,enormous size of higher eukaryotic introns,poses a major challenge to splice site recognition.As a consequence,in higher eukaryotes spliceosomes typically first assemble across exons(Robberson et al.,1990),which are rather short and homogeneous in size,and subsequently switch in various ways to a cross-intron configuration(Schneider et al.,2010).Initial identification of possible splice sites on a substrate commits a pre-mRNA to the splicing pathway,while it is only during subsequent cross-exon to cross-intron switching that particular splice sites are func-tionally paired and the ultimate splicing pattern of a pre-mRNA is cemented.Thus,initial cross-exon assembly also offers unique possibilities for regulating splice site choice.Choices between alternative splice sites are in part determined by trans-acting factors,which can be regulated in a development-,tissue-,differentiation-,or external stimulus-dependent manner(Kornblihtt et al.,2013).In particular,serine/arginine-rich proteins(SR proteins)and heterogeneous nuclear ribonucleoproteins(hnRNPs)typically promote or interfere with recruitment of the splicing machinery to particular splice sites,respectively.SR proteins and hnRNPs contain RNA-binding domains(typically one or several RNA recognition motifs RRMs),via which they bind to exonic/intronic splicing enhancer(ESE/ISE)sequences and exonic/intronic splicing silencer(ESS/ISS)sequences on pre-mRNAs,respectively(Zhu et al.,2001).SR proteins also contain an arginine-serine dipeptide-rich domain(RS domain),in which the serine residues can be reversibly phos-phorylated by SR protein kinases(SRPKs),thus modulating the RNA and protein interaction potentials of these domains(Zhou and Fu,2013).RS domains represent one example of intrinsically unstructured protein regions.Intrinsically unstructured proteins and protein regions are particularly abundant among splicing factors(Korneta and Bujnicki,2012),suggesting that many more regulatory principles of the spliceosome rely on them.They might enable the bridging of long distances,the contraction of distal components by folding-upon-binding transitions,fast binding kinetics,tunable interaction strengths or regulation by post-translational modifications.An additional level of complexity exists in many plants,fungi,and animals,which contain a second type of(minor or U12-dependent)spliceosome(Turunen et al.,2013).Although the minor spliceosome is responsible for the excision of less than 0.5%of introns in humans,which are characterized by distinct consensus sequences around the splice and branch sites,it is an essential machinery whose dysfunction is associated with diseases(Turunen et al.,2013).In the minor spliceosome,U11,U12,U4atac,and U6atac snRNPs functionally replace the major spliceoso-mal U1,U2,U4,and U6 snRNPs,respectively.U11 and U12 are composed of different snRNAs and proteins compared to U1 and U2,while U4atac and U6atac contain unique snRNAs but the same proteins as the corresponding U4 and U6 snRNPs.The U5 snRNP is part of both machineries.The minor spliceosome is thought to function according to similar principles as the major spliceosome,except that U11 and U12 snRNPs form a stable di-snRNP and assemble in one step.RefeRencesKornblihtt,A.R.,Schor,I.E.,All,M.,Dujardin,G.,Petrillo,E.,and Muoz,M.J.(2013).Nat.Rev.Mol.Cell Biol.14,153165.Korneta,I.,and Bujnicki,J.M.(2012).PLoS Comput.Biol.8,e1002641.Query,C.C.,and Konarska,M.M.(2006).Nat.Struct.Mol.Biol.13,472474.Robberson,B.L.,Cote,G.J.,and Berget,S.M.(1990).Mol.Cell.Biol.10,8494.Schneider,M.,Will,C.L.,Anokhina,M.,Tazi,J.,Urlaub,H.,and Lhrmann,R.(2010).Mol.Cell 38,223235.Semlow,D.R.,and Staley,J.P.(2012).Trends Biochem.Sci.37,263273.Turunen,J.J.,Niemel,E.H.,Verma,B.,and Frilander,M.J.(2013).Wiley Interdiscip Rev RNA 4,6176.Wahl,M.C.,Will,C.L.,and Lhrmann,R.(2009).Cell 136,701718.Zhou,Z.,and Fu,X.D.(2013).Chromosoma 122,191207.Zhu,J.,Mayeda,A.,and Krainer,A.R.(2001).Mol.Cell 8,13511361.