Control of Alternative Splicing by Signal-dependent Degradation of Splicing-regulatory Proteins

Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins. Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins. Alternative splicing is a fundamental mechanism operating in metazoan organisms to regulate protein expression (1Lynch K.W. Adv. Exp. Med. Biol... 2007; 623: 161-174Google Scholar, 2Shin C. Manley J.L. Nat. Rev. Mol. Cell Biol... 2004; 5: 727-738Google Scholar-3Stamm S. Hum. Mol. Genet... 2002; 11: 2409-2416Google Scholar). In humans and Drosophila, 30–95% of pre-mRNAs undergo alternative splicing, resulting in mature mRNAs that encode proteins with different sequences or that contain a premature stop codon and are subject to degradation (4Black D.L. Cell.. 2000; 103: 367-370Google Scholar, 5Brett D. Hanke J. Lehmann G. Hasse S. Delbruck S. Krueger S. Reich J. Bork P. FEBS Lett... 2000; 474: 83-86Google Scholar, 6Graveley B.R. Trends Genet... 2001; 17: 100-107Google Scholar, 7Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Amour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Science.. 2003; 302: 2141-2144Google Scholar, 8Kan Z. States D. Gish W. Genome Res... 2002; 12: 1837-1845Google Scholar, 9Mironov A.A. Fickett J.W. Gelfand M.S. Genome Res... 1999; 9: 1288-1293Google Scholar, 10Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Nat. Genet... 2008; 40: 1413-1415Google Scholar-11Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Nature.. 2008; 456: 470-476Google Scholar). Alternative splicing plays key roles in developmental processes, such as Drosophila sex determination, and defects in alternative splicing are commonly associated with human disorders, such as spinal muscular atrophy (12Förch P. Valcárcel J. Prog. Mol. Subcell. Biol... 2003; 31: 127-151Google Scholar, 13Singh N.N. Androphy E.J. Smith R.N. Crit. Rev. Eukaryotic Gene Expression.. 2004; 14: 271-285Google Scholar). Thus, understanding the molecular details of alternative splicing mechanisms has global implications for understanding both normal and disease states. At its basic level, the choice of splicing pattern for a pre-mRNA involves the regulation of spliceosome assembly on introns to be excised. Spliceosomes contain five small nuclear ribonucleoproteins (snRNPs) 3The abbreviations used are: snRNP, small nuclear ribonucleoprotein; CHX, cycloheximide; CPT, camptothecin; dsRNA, double-stranded RNA; GFP, green fluorescent protein; HA, hemagglutinin; hnRNP, heterogeneous ribonucleoprotein; IR, ionizing radiation; qPCR, quantitative real-time PCR; RNAi, RNA interference; RRM, RNA recognition motif; RS, arginine/serine-rich; SR, serine/arginine-rich; E3, ubiquitin-protein isopeptide ligase. (U1, U2, U4, U5, and U6) and numerous regulatory proteins, including the heterodimeric complex U2AF (U2 auxiliary factor) (14Black D.L. Annu. Rev. Biochem... 2003; 72: 291-336Google Scholar, 15Brow D.A. Annu. Rev. Genet... 2002; 36: 333-360Google Scholar-16Patel A.A. Steitz J.A. Nat. Rev. Mol. Cell Biol... 2003; 4: 960-970Google Scholar). Spliceosomes serve to define 5′ and 3′ splice sites at exon/intron boundaries and carry out the two transesterification reactions of splicing. Splicing-regulatory proteins that control interactions between general splicing complexes and pre-mRNA splice sites are central to the process of alternative splicing. Serine/arginine-rich (SR) proteins constitute a major class of splicing-regulatory proteins. All SR proteins contain one or two RNA recognition motifs (RRMs) and at least one arginine/serine-rich (RS) dipeptide domain (17Lin S. Fu X.D. Adv. Exp. Med. Biol... 2007; 623: 107-122Google Scholar). RRMs possess sequence-specific RNA binding activity, and, thus, can determine pre-mRNA substrate specificity, and RS domains mediate protein-protein interactions (17Lin S. Fu X.D. Adv. Exp. Med. Biol... 2007; 623: 107-122Google Scholar, 18Maris C. Dominquez C. Allain F.H. FEBS J... 2005; 272: 2118-2131Google Scholar). To illustrate, Tra2 (Transformer-2) is an evolutionarily conserved SR protein that contains N- and C-terminal RS domains and a central RRM. In Drosophila, Tra2 directs sex-specific alternative splicing of the dsx (doublesex) pre-mRNA. In vitro studies indicate that, through interactions with other SR proteins, Tra2 facilitates recruitment of general splicing complexes to female-specific regulatory sequences in the dsx pre-mRNA, resulting in utilization of an alternative 3′ splice site (19Qi J. Su S. McGuffin M.E. Mattox W. Nucleic Acids Res... 2006; 34: 6256-6263Google Scholar, 20Sciabica K.S. Hertel K.J. Nucleic Acids Res... 2006; 34: 6612-6620Google Scholar). In mammals, Tra2 orthologs direct tissue-specific alternative splicing of numerous pre-mRNAs, including those that encode proteins involved in breast cancer and sexual differentiation (21Venables J.P. Dalgliesh C. Paronetto M.P. Skitt L. Thorton J.K. Saunders P.T. Sette C. Jones K.T. Elliot D.J. Hum. Mol. Genet... 2004; 13: 1525-1534Google Scholar, 22Watermann D.O. Tang S. Res... 2006; Scholar). To mechanisms of alternative splicing, we splicing of the Drosophila TAF1 (TBP-associated factor 1) pre-mRNA in response to the drug camptothecin an inhibitor of Nat. Rev. 2006; Scholar, M.S. D.A. Mol. Biol... 2006; Scholar). CPT the alternative splicing of other in Drosophila and in humans M.S. D.A. 2008; 14: Scholar, S. J. J. J. C. L. L. Mol. Res... 2004; Scholar). TAF1 the of the general factor plays a central in the of Biol... 2002; 12: Scholar, C. Nature.. 2005; Scholar, O. R. S. Adv. 2004; R. Nature.. Scholar). Alternative splicing of the Drosophila TAF1 pre-mRNA involves the of and and TAF1 and M.S. D.A. Mol. Biol... 2006; Scholar). and an binding binding to and protein D.A. J. 2006; Scholar). TAF1 are in Drosophila the of tissue-specific mechanisms of alternative splicing D.A. J. 2006; Scholar, D.A. 2007; Scholar). and splicing is in Drosophila S2 cells in response to CPT M.S. D.A. Mol. Biol... 2006; Scholar). of and splicing by CPT is by the response ATR (ATM-RAD3-related) and the In as indicated by studies in the of TAF1 splice sites was of splicing involves recruitment of general splicing complexes to splice sites associated with and M.S. D.A. 2008; 14: Scholar). on we that splicing-regulatory proteins function of the CPT-induced signaling pathway to mediate the change in TAF1 alternative splicing. we the of a screen for TAF1 pre-mRNA alternative splicing-regulatory proteins and a screen to targets of the CPT-induced signaling of one of the splicing-regulatory proteins, that the ATR pathway TAF1 alternative splicing by signaling Tra2 and of for the screen was out and by J.W. B.R. 2004; Scholar). in and and by to for and to was as by M.S. D.A. Mol. Biol... 2006; Scholar). the TAF1 by of as in M.S. D.A. Mol. Biol... 2006; Scholar). of the that is the TAF1 by qPCR, as by M.S. D.A. Mol. Biol... 2006; Scholar). as 5′ to and and The of was to by the Nucleic Acids Res... 2001; is an and is the for to the an independent of of cells used as independent of and was used when multiple that Tra2 or by a a expression Nucleic Acids Res... Scholar). the 5′ to and and of the that was as for M.S. D.A. Mol. Biol... 2006; Scholar). of was the and by The green fluorescent protein expression was by an S. J.W. 2000; Cell S2 cells at in Drosophila and cells to a of in of in cells with of and of was to the and an cells a of CPT in to the indicated as a an of at for the indicated cells with CPT and in in in or MG132 in ionizing cells with of a and to at for the indicated experiments by two different was an independent and and and or was an a and and is for CPT-induced down-regulation of of expression in cells with or CPT in the or of for the indicated of protein molecular are indicated on the by the was used as a of expression in the of as for in of ATR in or ATR cells with or CPT of expression in or ATR with or CPT for is for CPT-induced down-regulation of of expression in cells with or CPT in the or of for the indicated of protein molecular are indicated on the by the was used as a of expression in S2 cells with to expression in the or of S2 cells by and in of by to with by S. of P. R. O. S. Hum. Mol. Genet... 2004; 13: or D.A. Mol. Biol... 2002; and by the The human used to is in to Drosophila Tra2 at of are of S2 cells was out as by D.A. Mol. Biol... 2002; Scholar). of cells and was two was with for and the other was with CPT for The was an and the was with with and cells at the same an for of TAF1 Alternative identify proteins that control and alternative splicing in Drosophila S2 we an screen by J.W. B.R. 2004; Scholar). in vitro was used to expression of of the splicing and RNA binding proteins. S2 cells with for RNA was the and TAF1 and used to TAF1 between and cells that a on TAF1 in independent experiments by was used to determine and levels. The screen identified 15 in S2 cells was of and splicing, the same change in TAF1 alternative splicing that occurs in response to CPT 1) M.S. D.A. Mol. Biol... 2006; Scholar). was for of the 15 TAF1 by that the in and was to a change in the of TAF1 of the are for Tra2 and in and and of was M.S. D.A. Mol. Biol... 2006; of and splicing of and is on to the to cells with of and CPT on and splicing Tra2 of and is on to the to cells of and CPT on and in a new of CPT-induced and determine of the 15 encode targets of the CPT-induced signaling we used to and in S2 cells that with and We that a protein of CPT, the treatment and to with that of treatment or CPT treatment In a protein independently of CPT, the treatment and to with the of treatment and CPT treatment. cells with CPT for to a in and M.S. D.A. Mol. Biol... 2006; Scholar). reduced Tra2 to of cells the of Tra2 in a in and levels. However, CPT treatment of Tra2 cells or to Tra2 and for and 1) M.S. D.A. Mol. Biol... 2006; Scholar). Tra2 and the other five proteins in the CPT-induced signaling pathway and that the pathway the function of proteins. we to proteins as splicing-regulatory proteins. reduced to of cells In to CPT treatment of Tra2 CPT treatment of cells and to and for the proteins Thus, and the proteins function independently of splicing-regulatory proteins to regulate TAF1 alternative splicing. of the splicing-regulatory proteins, reduced the of the and However, to the of the mechanism with was that the in and splicing was to To this we used to and in S2 cells with for of the splicing-regulatory proteins. In the and suggesting that are for the change in TAF1 alternative splicing 1) CPT of and on studies of other splicing-regulatory proteins, a CPT-induced TAF1 alternative splicing by the or function of splicing-regulatory proteins. in the of SR proteins, is subject to regulation by small in protein alternative splicing and of the RS domains protein-protein interactions (17Lin S. Fu X.D. Adv. Exp. Med. Biol... 2007; 623: 107-122Google Scholar, J. Su S. McGuffin M.E. Mattox W. Nucleic Acids Res... 2006; 34: 6256-6263Google Scholar, 22Watermann D.O. Tang S. Res... 2006; Scholar). To the CPT-induced regulatory mechanism, we the of S2 cells with Tra2 and, as a expression was by the of to the for or CPT was to the and, for was by of an and a that to the of control cells In when was in cells a was the was in the and of both and of that a CPT-induced To this of was used to determine and expression levels. In of the this that and expression was reduced of CPT treatment and was in the of at to was at In expression of or in with was reduced by CPT that down-regulation of or was to the or of the expression Additionally, of proteins by a protein pattern in and cells the of to the CPT-induced protein down-regulation mechanism is for CPT-induced down-regulation of the of the Tra2 used in the that to arginine are indicated by is the and between the and indicated by of or lysine in S2 cells with or CPT for by the was used as a of protein molecular are indicated on the with the that Tra2 directly regulates TAF1 alternative splicing, the and of CPT-induced down-regulation of expression was to the and of CPT-induced of and splicing and M.S. D.A. Mol. Biol... 2006; Scholar). that a CPT-induced targets and for by the of down-regulation of S2 cells to We found that the pathway to the same change in TAF1 alternative splicing as CPT M.S. D.A. Mol. Biol... 2006; Scholar). However, expression was of of IR, splicing was of TAF1 alternative splicing is by the ATR signaling pathway in response to CPT and the signaling pathway in response to IR, indicate that different signaling pathways that a change in TAF1 alternative splicing different splicing-regulatory proteins. conclusion is by the that only a subset of identified in the screen function of CPT CPT of determine the to Tra2 expression is by CPT-induced we used an human to Drosophila Tra2 P. R. O. S. Hum. Mol. Genet... 2004; 13: Scholar). of S2 the Tra2 a protein of the and of Tra2 caused a in the of the protein to a as the in the of the and and the of the of S2 cells with or CPT for that the of Tra2 was reduced by CPT treatment the of CPT-induced Tra2 was to the of Tra2 with the that Tra2 is to and splicing. Additionally, the of the on Tra2 expression was to that of Thus, to the CPT Tra2 is a of Tra2 of CPT in S2 in down-regulation of and expression occurred the of CPT, suggesting that the regulatory mechanism new protein To this S2 cells with the protein inhibitor and by In the of CPT, treatment on of that the is to CPT-induced down-regulation of S2 cells with CPT and inhibited new protein CPT-induced down-regulation of expression indicate that down-regulation new protein synthesis, is with that CPT-induced of and splicing new protein M.S. D.A. Mol. Biol... 2006; Scholar). ATR for CPT-induced of determine the of the ATR signaling pathway in CPT-induced down-regulation of we small the of such as and at the of ATR at 2004; Scholar, P. Res... 1999; Res... Scholar). S2 cells that with CPT, or both CPT and and by that inhibited CPT-induced down-regulation of that is involved in the regulatory mechanism S2 cells with CPT, or both CPT and However, CPT-induced down-regulation of suggesting that is involved in the mechanism To directly the for ATR in CPT-induced down-regulation of was used to ATR expression in S2 the of the S2 cells with ATR dsRNA, on the the cells with on the expression was with and on the the cells with CPT for and that reduced ATR in both the and of CPT and inhibited CPT-induced down-regulation of expression and the inhibitor and indicate that ATR is for CPT-induced down-regulation of the same signaling pathway regulates expression and TAF1 alternative splicing, is that Tra2 directly regulates TAF1 alternative splicing. CPT-induced of down-regulation of expression by mechanisms that or are the same was used to of both and only expression was by CPT treatment and are of new protein by was to down-regulate expression in Thus, we that CPT post-translational we that CPT and proteasome-dependent degradation of splicing-regulatory proteins R. Trends Cell Biol... 2004; 14: Scholar, Trends Cell Biol... Scholar). The is for the of protein degradation in and has in the response to by CPT R. Trends Cell Biol... 2004; 14: Scholar, C. Res... 2007; Scholar, Luo Zhang J. D. J. J. 2007; R. J. 2008; Scholar). To the to the proteasome is involved in CPT-induced down-regulation of S2 cells with the proteasome inhibitor MG132 Trends Cell Biol... Scholar). that MG132 inhibited CPT-induced down-regulation of expression S2 cells with CPT and MG132 of indicate that is by the proteasome in response to a CPT-induced the of in the degradation mechanism. proteasome-dependent degradation of splicing-regulatory proteins is a of the CPT signaling pathway that and splicing, proteasome be to CPT-induced and splicing. To this TAF1 in S2 cells with CPT or MG132 to cells with CPT and that MG132 TAF1 alternative splicing, MG132 inhibited CPT-induced of and splicing and indicate that of and splicing proteasome-dependent degradation of splicing-regulatory proteins. of a in CPT-induced on of the proteasome in CPT-induced degradation, we that of a lysine in is of the degradation mechanism R. Trends Cell Biol... 2004; 14: Scholar). To determine the to the lysine in Drosophila Tra2 and to the degradation mechanism, we the expression of proteins in to is to lysine is a substrate for S2 cells proteins with as a or CPT for and by to expression of the and was in cells with CPT for In the was in cells with CPT for at in cells with for indicate that post-translational of in occurs in response to CPT and signals proteasome-dependent for the of Tra2 was by of the Drosophila Tra2 protein with Tra2 proteins other including those and that and the other lysine are evolutionarily conserved is to that such as contain two Tra2 one gene a protein with lysine at to and and the other gene that in to is for Tra2 as a site for post-translational and that in expression of Tra2 that can for Alternative the screen used to identify of TAF1 alternative splicing has used to identify of other alternative splicing J.W. B.R. 2004; Scholar, S. J. J.M. B.R. Nat. Mol. Biol... 2007; 14: Scholar). In with that TAF1 alternative splicing encode of the spliceosome and and and a and an protein J.W. B.R. 2004; Scholar). Thus, are two basic of alternative splicing proteins. of the are for and by U2AF function to regulate different of alternative splicing. is with the that in roles in splicing and that of in human cells the alternative splicing pattern of a subset of Mol. Biol... 2006; 17: Scholar, J.A. C. Biol... 2007; 5: Scholar). In of the by regulate alternative splicing of both are targets of the CPT-induced signal, suggesting that the two function to regulate alternative splicing. of the splicing the TAF1 screen as the that gene of the alternative splicing and only a alternative splicing that global on alternative splicing of the that the identified of independent between To illustrate, is between that TAF1 alternative splicing and that regulate alternative 5′ splice site utilization or that regulate of Thus, different of alternative splicing a of regulatory proteins. for TAF1 Alternative by Splicing-regulatory the was that the screen identify of alternative such as heterogeneous ribonucleoproteins that function to SR proteins through binding splicing (14Black D.L. Annu. Rev. Biochem... 2003; 72: 291-336Google Scholar, S. Fu X.D. Adv. Exp. Med. Biol... 2007; 623: 107-122Google Scholar, Valcárcel J. Trends 2000; Scholar, R. P. L. Adv. Exp. Med. Biol... 2007; 623: Scholar). of assembly of splicing complexes at alternative splice sites and alternative However, and splicing by of The screen of the Drosophila of and alternative splicing when knocked by in S2 cells J.W. B.R. 2004; Scholar). of the splicing-regulatory proteins identified in the screen are characterized as of Tra2 is an SR protein that facilitates sex-specific recognition of alternative splice sites in Drosophila by binding splicing and, through with the RS U2AF or the to splicing elements K.S. Hertel K.J. Nucleic Acids Res... 2006; 34: 6612-6620Google Scholar, Cell.. Scholar). is an protein of protein-protein In facilitates interactions with the spliceosome S. 1999; 5: Scholar). and are proteins that contain RRMs S.F. S. S. Gene Scholar). In proteins bind sequences and splicing of that contain alternative with 5′ splice sites by P. Valcárcel J. 2001; Scholar). and are of the spliceosome that function in 3′ splice site recognition M.P. 2007; Scholar, I. J. FEBS J... 2006; Scholar). is a that the 3′ splice site and is a that the are for how the of Tra2 or other splicing-regulatory proteins of TAF1 alternative and Tra2 bind splicing to alternative In of this mechanism, Tra2 splicing of the its pre-mRNA in a In Drosophila Tra2 by binding an splicing and with spliceosome assembly J. Mattox W. Mol. Biol... 2003; Scholar). in Drosophila Tra2 are and the is the of Tra2 is to Thus, in the of TAF1 alternative splicing, Tra2 bind splicing in introns and to of and the CPT-induced in Tra2 this binding sites for Tra2 in the TAF1 pre-mRNA an evolutionarily conserved splicing is in the between and M.S. D.A. 2008; 14: Scholar). Tra2 bind an splicing in to splicing between and resulting in alternative and Tra2 splicing complex assembly at splice complexes at and splice sites to for with and splice The U2AF and be to this mechanism, alternative splicing can be by regulation of U2AF on the pre-mRNA M.P. 2007; Scholar, 2008; Scholar). with this the 3′ splice site to the to the 3′ splice site of M.S. D.A. 2008; 14: Scholar). In this the splicing-regulatory proteins function to define splice of one of the proteins by is to and indicate that the of splicing-regulatory proteins in a can pre-mRNA alternative splicing of Splicing-regulatory Proteins Alternative indicate that TAF1 alternative splicing in response to CPT ATR proteasome degradation of splicing-regulatory proteins. In to the proteasome-dependent degradation of Tra2 and in response to CPT, for a for the proteasome in alternative splicing that a proteasome inhibitor CPT-induced TAF1 alternative splicing in S2 cells in Drosophila cells with the in proteasome to splicing R. Q. 2004; Scholar). The of a for the proteasome in splicing in the that alternative splicing is in degradation of splicing-regulatory proteins is J... 2003; in cells that in response to of RNA by is at the RS to nuclear and by the proteasome of the SR protein In this proteasome-dependent degradation of other SR proteins, and the degradation involves a C-terminal RS domain and an J.P. Dalgliesh C. L. J. Elliot D.J. Hum. Mol. Genet... 2005; 14: in cells that a splicing-regulatory protein is for proteasome-dependent degradation by the ubiquitin and that alternative splicing is by J. 2005; that treatment of human cells proteasome-dependent degradation of Thus, that the of splicing-regulatory proteins by proteasome-dependent degradation be a mechanism for alternative splicing in response to or of Splicing-regulatory indicate that activation of the ATR signaling pathway by CPT treatment of S2 cells proteasome-dependent degradation of resulting in of and splicing. The that between ATR activation and Tra2 degradation to be The that protein is for CPT-induced Tra2 degradation the of post-translational in the degradation mechanism. protein degradation by the proteasome involves protein by Mol. Cell.. 2007; Scholar). In of is direct that Tra2 is to CPT-induced Tra2 function in alternative splicing is by of the RS domains and by a direct with protein (17Lin S. Fu X.D. Adv. Exp. Med. Biol... 2007; 623: 107-122Google Scholar, Tang M.E. C. P. L. S. Hum. Mol. Genet... 2008; 17: Scholar). However, is in response to CPT, contain an RS domain or a protein binding is that and other splicing-regulatory proteins are in a and degradation of Tra2 in of the complex and degradation of the proteins. In of proteasome by MG132 to the of of Tra2 in the of in the degradation mechanism is that MG132 an that occurs to and is for Tra2 such as ATR In proteasome of and ATR in response to C. Res... 2007; Scholar, Luo Zhang J. D. J. J. 2007; R. J. 2008; Scholar). the other is by the of lysine other lysine was to CPT-induced degradation, suggesting that of is for degradation The of be to of an alternative lysine or to a mechanism S. 2003; Scholar). In degradation of splicing-regulatory proteins be a general mechanism to regulate alternative splicing. in be to identify signaling and involved in post-translational of splicing-regulatory proteins that mediate TAF1 alternative splicing. We are to J. and for the and for the screen and to C. and S. for the Tra2 with