Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3

Abstract

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3′ splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3′ splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes. Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3′ splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3′ splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes. The spliceosome is the large and dynamic macromolecular complex responsible for removing intron sequences that interrupt many eukaryotic gene transcripts. Spliceosomes are composed of five small nuclear ribonucleoproteins (snRNPs), 2The abbreviations used are:snRNPsmall nuclear ribonucleoproteinRG-richarginine- and glycine-richGSTglutathione S-transferaseY2Hyeast two-hybrid. known as U1, U2, U4, U5, and U6, and more than 100 additional proteins (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). In vitro splicing studies show that snRNPs and additional protein factors come together with a pre-mRNA transcript in an ordered assembly via short-lived intermediate complexes to carry out the chemistry of splicing (2Moore M.J. Query C.C. Sharp P.A. Gesteland R. Atkins J. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 333-360Google Scholar). Precisely how the assembly of these larger complexes is executed and controlled and which factors are responsible are poorly understood. small nuclear ribonucleoprotein arginine- and glycine-rich glutathione S-transferase yeast two-hybrid. Much of our knowledge about the identity and function of individual spliceosome proteins is the result of extensive genetic and biochemical analyses in the yeast Saccharomyces cerevisiae (3Brow D.A. Annu. Rev. Genet. 2002; 36: 333-360Crossref PubMed Scopus (294) Google Scholar, 4Burge C.B. Tuschl T. Sharp P.A. Gesteland R.F. Cech T.R. Atkins J.F. 2nd Ed. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 525-560Google Scholar). Although the yeast spliceosome is fundamentally similar to the mammalian spliceosome, the mammalian spliceosome must handle more complex splicing substrates and is subject to more complex regulation. Therefore, it is likely that some mammalian splicing factors have no functional correlate in yeast. Consistent with this idea, recent analyses of human spliceosomes by mass spectrometry identified a significant number of proteins not previously associated with splicing (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 5Neubauer G. King A. Rappsilber J. Calvio C. Watson M. Ajuh P. Sleeman J. Lamond A. Mann M. Nat. Genet. 1998; 20: 46-50Crossref PubMed Scopus (422) Google Scholar, 6Behzadnia N. Golas M.M. Hartmuth K. Sander B. Kastner B. Deckert J. Dube P. Will C.L. Urlaub H. Stark H. Luhrmann R. EMBO J. 2007; 26: 1737-1748Crossref PubMed Scopus (161) Google Scholar, 7Deckert J. Hartmuth K. Boehringer D. Behzadnia N. Will C.L. Kastner B. Stark H. Urlaub H. Luhrmann R. Mol. Cell. Biol. 2006; 26: 5528-5543Crossref PubMed Scopus (228) Google Scholar). Many of these proteins do not have an obvious homolog in S. cerevisiae, and subsequent in vitro studies demonstrated a critical role for some of these factors in splicing (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 10Lallena M.J. Chalmers K.J. Llamazares S. Lamond A.I. Valcarcel J. Cell. 2002; 109: 285-296Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). A major challenge is to determine the specific functions of these specialized mammalian splicing factors. Human SPF30 (also known as SMNrp) protein was first identified by mass spectrometry as a component of purified spliceosomes (5Neubauer G. King A. Rappsilber J. Calvio C. Watson M. Ajuh P. Sleeman J. Lamond A. Mann M. Nat. Genet. 1998; 20: 46-50Crossref PubMed Scopus (422) Google Scholar) and initially characterized as a nuclear protein containing a Tudor domain, a motif found in RNA-binding proteins implicated in binding methylated arginines (11Talbot K. Miguel-Aliaga I. Mohaghegh P. Ponting C.P. Davies K.E. Hum. Mol. Genet. 1998; 7: 2149-2156Crossref PubMed Scopus (63) Google Scholar). Although the sequence of SPF30 protein is generally conserved within higher eukaryotes, there is no S. cerevisiae homolog. SPF30 does share ∼51% sequence identity within the Tudor domain of SMN (survival of motor neuron), a protein important for snRNP biogenesis (40Fischer U. Liu Q. Dreyfuss G. Cell. 1997; 90: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Mutations in SMN are believed to be responsible for its role in the autosomal recessive disease spinal muscular atrophy (12Campbell L. Potter A. Ignatius J. Dubowitz V. Davies K. Am. J. Hum. Genet. 1997; 61: 40-50Abstract Full Text PDF PubMed Scopus (272) Google Scholar). The Tudor domain of SMN is sufficient for binding four of the Sm/Lsm proteins that make up a core heptamer of the U snRNPs (SmB, SmD1, SmD3, and Lsm4). These four proteins contain arginine- and glycine-rich (RG-rich) motifs, and SMN protein has been shown to bind preferentially to dimethylarginine-modified residues within the RG-rich repeats of SmD1, SmD3 (13Friesen W.J. Massenet S. Paushkin S. Wyce A. Dreyfuss G. Mol. Cell. 2001; 7: 1111-1117Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In addition, the Tudor domains of both SMN and SPF30 have been shown to preferentially bind a synthetic symmetrically dimethylated (RsDMAG)4 peptide identical to a sequence within the C terminus of the human SmD1 protein (14Cote J. Richard S. J. Biol. Chem. 2005; 280: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). A critical role for SPF30 in splicing was demonstrated in a human in vitro system. Immunodepletion of the SPF30 protein from human nuclear cell extracts blocks spliceosome assembly on a minimal splicing substrate at the transition from prespliceosomes (A complex) to assembled spliceosomes (B complex) but does not appear to be necessary for tri-snRNP assembly (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Additionally, SPF30 was identified by mass spectrometry in purified human prespliceosomes and spliceosomes but was not detected in spliceosomes activated for catalysis in which the U4 and U1 snRNP association is complex) N. Golas M.M. Hartmuth K. Sander B. Kastner B. Deckert J. Dube P. Will C.L. Urlaub H. Stark H. Luhrmann R. EMBO J. 2007; 26: 1737-1748Crossref PubMed Scopus (161) Google Scholar, 7Deckert J. Hartmuth K. Boehringer D. Behzadnia N. Will C.L. Kastner B. Stark H. Urlaub H. Luhrmann R. Mol. Cell. Biol. 2006; 26: 5528-5543Crossref PubMed Scopus (228) Google Scholar, K. Urlaub H. Will C.L. M. M. Luhrmann R. U. S. A. 2002; PubMed Scopus Google Scholar, M.S. N. Moore M.J. RNA 2002; PubMed Scopus Google Scholar, Urlaub H. M. Will C.L. M. Luhrmann R. 2002; PubMed Scopus Google Scholar). The prespliceosome to spliceosome transition is characterized by the addition of the tri-snRNP to a complex of U1 and U2 snRNPs to the splice site and of a pre-mRNA SPF30 has been shown to with both the U2 snRNP and the suggesting that it may as a between the prespliceosome and tri-snRNP (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A protein interaction between SPF30 and a U4/U6 di-snRNP was (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar). the its specific interaction and by which SPF30 as this potential is Here we and interactions to individual protein a in which specific domains of SPF30 interact with in both the prespliceosome and the potentially together within the we note that these interactions are not conserved in yeast, suggesting may be important for the more complex splicing of found in and and from cell RNA proteins in purified by glutathione by and of snRNPs from of purified protein was to nuclear PubMed Scopus Google Scholar) in was the has been observed that tri-snRNP complex can be U. Luhrmann R. R. A. PubMed Scopus Google we used nuclear to of spliceosome snRNPs from the a from to RNA was purified from and and by was out U1, U2, U4, U5, and M.M. Sharp P.A. Cell. Full Text PDF PubMed Scopus Google Scholar). from RNA the and yeast with and cell from yeast and by with to that and protein was and of the of the of and and yeast on in and to for interaction between the and of in vitro proteins in vitro in and to glutathione to of purified These to with for at by in containing The glutathione was in of and and the by and from and out as previously M. E. G. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). of nuclear was with of for at in and The was in the with by an containing was 100 and and at The of U2AF35 in extracts and was by U2AF35 of SPF30 with is a critical splicing factor in human extracts and interacts with spliceosome snRNPs (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). the role of SPF30 in we the snRNP interactions are and by different domains of SPF30. of purified SPF30 protein and in protein we domains of SPF30 not We and purified of these domains from and protein We the association of the SPF30 domains with snRNPs by an in vitro binding in which we nuclear from different SPF30 domains to we with a and detected U in by We also show by that the snRNP protein is in the the that is of the snRNP interactions with spliceosome for snRNP core protein in nuclear from and from a for spliceosome U in nuclear from and from a for spliceosome U from to of nuclear from to N-terminal Tudor C-terminal studies that SPF30 U4 and snRNPs and to a U2 and snRNPs from nuclear (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). no U1 association was In we five U2, U4, U5, and in the with SPF30 SPF30 the snRNPs in a The interaction is with U4 and which both at is likely that the of U4 and a U4/U6 snRNP in a that with the of U4 and U6, likely as a of tri-snRNP. U1 and U2 snRNPs show a with both at with we association and We that SPF30 interacts with of the but with the SPF30 domains responsible for the snRNP we this with of to domains of SPF30. N-terminal of SPF30 U2 and U1 snRNPs the central Tudor domain of SPF30 U1 snRNP a C-terminal domain U4 and snRNPs are to a snRNP SPF30 the Tudor domain U2, U4, and snRNPs and to have minimal interactions with U1 and These show that domains of SPF30 are responsible for with different The N-terminal domain with both U2 and U1 with a complex that both The C-terminal domain interacts with U4 and likely as a U1 and be to SPF30 domains in a U1 interacts with the Tudor domain of SPF30 but also to interact with the N-terminal domain in some U2 snRNP. We association with likely as a of the tri-snRNP. SPF30 with in the and identify which protein factors interact with we out a yeast with U1, U2, U4, and this we identified several proteins that potentially interact with SmD1, SmD3, the protein hPrp3 U2AF35, a protein factor in 3′ splice site recognition and prespliceosome assembly B. Cell. Full Text PDF PubMed Scopus Google Scholar, L. S. D. C. Valcarcel J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, D.A. T. PubMed Scopus Google Scholar) and The interaction with hPrp3, which is a component of both the U4/U6 di-snRNP and was not studies that in vitro binding of this protein with SPF30 (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar). the interactions with in particular, U2AF35 are these interactions and to specific domains within the we both the and in vitro as C terminus of SPF30 interacts with the middle domain of of SPF30 with of SPF30 and in vitro terminus of SPF30 interacts with the N-terminal domain of of SPF30 with of SPF30 and in vitro SPF30 and in vitro U2AF35 and The SPF30 Tudor to with SmD1, SmD3, and with SmD1 and SmD3, we both the and C-terminal domains of these proteins identified in a of proteins C. S. R. E. Luhrmann R. J. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The N-terminal domain of SmD1 and SmD3 the and for the C-terminal domain RG-rich repeats that can be observed by the central Tudor domain of SPF30 is sufficient to interact with SmD1, SmD3, and Lsm4 and both the and the Tudor domain of SPF30 interact with the C-terminal domains of both SmD1 and In of the C-terminal from SmD1 SmD3 no A and These that SPF30 interacts with a of the Sm/Lsm studies that the Tudor domain of SPF30 can bind an RG-rich containing (14Cote J. Richard S. J. Biol. Chem. 2005; 280: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (193) Google it is likely that the Sm/Lsm protein interactions with SPF30 are by its Tudor domain and the RG-rich C-terminal tails of SmD1, SmD3, and of the Sm/Lsm SmD1, SmD3, and Lsm4 contain extensive RG-rich C-terminal the interaction between these we in vitro SPF30 and in vitro SmD1, SmD3, and we proteins in the and no protein in with and These a interaction of the SPF30 Tudor domain with the RG-rich Sm/Lsm of SPF30 with the U4/U6 hPrp3 and the U2 snRNP the interaction of SPF30 with hPrp3, we several hPrp3 domains domain for hPrp3 previously in a protein interactions between hPrp3 and A. J. C. Liu J. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We a hPrp3 is with SPF30 but not with SPF30 Tudor domain of the hPrp3 domains containing a with both SPF30 and but not these central These that it is the central of hPrp3 that interacts with SPF30. a we and in vitro the the C-terminal domain of we in vitro hPrp3 in observed in the yeast hPrp3 proteins containing residues but not these are by SPF30. does not of the hPrp3 protein These show that the C-terminal domain of SPF30 is sufficient to interact with the central domain of the U2 snRNP proteins we in the U2AF35 a We used an similar to that for hPrp3 to the of U2AF35 and SPF30 that interact with the N-terminal and C-terminal domains of U2AF35 on studies of the protein C.L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google we for interactions with SPF30. SPF30 and Tudor show with the N-terminal domain of U2AF35 result that the N-terminal domain of U2AF35 interacts with SPF30 residues of the Tudor a we in vitro N-terminal domain SPF30. We both in vitro U2AF35 and containing the N-terminal domain residues and residues the of which the motif of U2AF35 U2AF35 is observed with U2AF35 as a with we also SPF30 interacts with U2AF35 in the of We that SPF30 does not interact with by yeast by in vitro binding not it can both U2AF35 and in an in vitro binding these show that the N-terminal domain of SPF30 is sufficient to interact with the N-terminal domain of U2AF35 to the SPF30 with U2 nuclear extracts SPF30 protein previously was shown by to with the U2 snRNP but not U4/U6 di-snRNP tri-snRNP (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar). to the SPF30 interaction with U2AF35, a U2 snRNP associated is sufficient to mediate the SPF30 association with U2 snRNP. these we nuclear extracts of U2AF35 protein B. Cell. Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). extracts of both and U2AF35 for the subsequent of the We nuclear extracts with N-terminal domain to glutathione in the of purified the N-terminal domain of SPF30 U1 and U2 snRNPs from extracts of U2AF35 SPF30 association with U2 snRNP is not in extracts as observed by not SPF30 association with U1 snRNP observed in these to be These that additional to U2AF35 the SPF30 association with U2 snRNP. SPF30 an between U2AF35 and studies that SPF30 may as between prespliceosomes and thereby spliceosomes to (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In this we identified and characterized interactions via domains of SPF30 with both U2AF35 and proteins associated with prespliceosomes and these we SPF30 can with both U2AF35 and hPrp3 We protein to glutathione and with U2AF35 in the of SPF30. in the of SPF30 not U2AF35, in the of SPF30 the of U2AF35 These that SPF30 may an interaction between hPrp3, a core tri-snRNP with U2AF35, an splicing factor in Spliceosome assembly is a complex and dynamic process involving a of proteins and Although a number of specific interactions that mediate spliceosome assembly and function have been by genetic studies in S. a large to be In particular, the interactions that tri-snRNP association with spliceosomes are not understood. there are likely many additional interactions by the more complex splicing in more complex eukaryotes. In this we on the human protein SPF30 and its interactions with spliceosome We that SPF30 interacts with five U SPF30 we specific snRNP interactions to domains of SPF30. The N-terminal domain of SPF30 interacts with U2 and U1 The Tudor domain to interact with U1 the C-terminal domain interacts with the U4/U6 Importantly, these with the specific snRNP proteins that we found interact with SPF30 The N-terminal domain of SPF30 U2AF35, a protein associated with the prespliceosome and U2 snRNP. We also that SPF30 interacts with U2AF35 in the of suggesting that SPF30 interacts with an this interaction is not sufficient to SPF30 association with U2 snRNP. studies on U2AF35 on its role in spliceosome B. Cell. Full Text PDF PubMed Scopus Google Scholar, L. S. D. C. Valcarcel J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, D.A. T. PubMed Scopus Google Scholar). U2AF35 interacts with its via residues M. M. Lamond A.I. U. S. A. PubMed Scopus Google Scholar). the the pre-mRNA and 3′ splice has been shown to interact with a component of U2 and this interaction may the recruitment and association of U2 snRNP with the pre-mRNA 2006; PubMed Scopus Google Scholar, R. U. O. Will C. R. G. L. Luhrmann R. M. RNA 2006; 12: PubMed Scopus Google Scholar). SPF30 interaction with U2AF35 may recruitment and association of tri-snRNP with the prespliceosome. U2AF35 is in assembled its role at this of assembly is not Urlaub H. M. Will C.L. M. Luhrmann R. 2002; PubMed Scopus Google Scholar). is to that may play a critical role in both prespliceosome and the transition to assembled The C-terminal domain of SPF30 interacts with hPrp3 is a component of the U4/U6 di-snRNP and and S. R. Luhrmann R. RNA 2006; 12: PubMed Scopus Google Scholar). to the spliceosome with tri-snRNP the of complex Urlaub H. M. Will C.L. M. Luhrmann R. 2002; PubMed Scopus Google Scholar). is that tri-snRNP recruitment to the spliceosome is in by the interaction of the C-terminal domain of SPF30 and SPF30 is from spliceosomes at prespliceosomes as observed previously (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar, 9Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can both (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google Scholar) and Rappsilber J. Ajuh P. Lamond A.I. Mann M. J. Biol. Chem. 2001; 276: 31142-31150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) that SPF30 as a between U2 snRNP and tri-snRNP at the transition to assembled this and a potential SPF30 may function as a between 3′ splice site recognition in the prespliceosome and recruitment of the tri-snRNP more studies are to this function is also that and binding are on mass spectrometry of purified splicing SPF30 to the spliceosome at prespliceosome assembly and to tri-snRNP addition N. Golas M.M. Hartmuth K. Sander B. Kastner B. Deckert J. Dube P. Will C.L. Urlaub H. Stark H. Luhrmann R. EMBO J. 2007; 26: 1737-1748Crossref PubMed Scopus (161) Google Scholar, K. Urlaub H. Will C.L. M. M. Luhrmann R. U. S. A. 2002; PubMed Scopus Google Scholar). we a U4/U6 di-snRNP association with SPF30 in In SPF30 in nuclear extracts associated with U2 snRNP and not U4/U6 di-snRNP tri-snRNP. These that there may be a to the association of SPF30 with spliceosome both U1 and U4 snRNP association with the spliceosome is as with U4 for U2 and the splice the hPrp3, and SPF30 appear to the of these proteins identified in activated spliceosomes (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, D. Sander B. Kastner B. Luhrmann R. Stark H. Nat. Mol. Biol. PubMed Scopus Google Scholar). potential role for SPF30 in the of the spliceosome to be The Tudor domain of SPF30 interacts with the RG-rich C-terminal tails of core snRNP proteins SmD1 and interaction likely at in on of RG-rich repeats in the of an was shown to the interaction of the SMN and SPF30 Tudor domain with this it is not (13Friesen W.J. Massenet S. Paushkin S. Wyce A. Dreyfuss G. Mol. Cell. 2001; 7: 1111-1117Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, J. Richard S. J. Biol. Chem. 2005; 280: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Lsm4 also RG-rich repeats known to similar to that of both SmD1 and SmD3 H. L. V. Fischer U. Luhrmann R. RNA 2001; 7: PubMed Scopus Google Scholar). is likely the SPF30 Tudor interaction with Lsm4 via the C-terminal domain of this to be A role for SPF30 interaction with the Sm/Lsm proteins in spliceosome assembly is more to these proteins are core of the is that the SPF30 Tudor association with U1 snRNP we by in vitro an interaction important for SPF30 association with this interaction with the U2AF35 interaction as a for the recruitment of tri-snRNP to a splicing the the interaction observed between SPF30 Tudor domain and U1 snRNP may be to the of the heptamer of U1 the Sm/Lsm heptamer of U2, U4, U5, and snRNPs may be in assembled nuclear the of the SPF30 Tudor domain to preferentially bind SmD1, SmD3, and Lsm4 may an additional role in nuclear snRNP The Tudor domain of SPF30 identity to SMN which an important role in snRNP biogenesis by the proteins the snRNPs L. J. Dreyfuss G. 2002; PubMed Scopus Google Scholar). SMN also to and Lsm4 via its Tudor domain H. L. V. Fischer U. Luhrmann R. RNA 2001; 7: PubMed Scopus Google Scholar). SPF30 with SMN protein at (8Meister G. Hannus S. Plottner O. Baars T. Hartmann E. Fakan S. Laggerbauer B. Fischer U. EMBO J. 2001; 20: 2304-2314Crossref PubMed Scopus (54) Google a site of snRNP and assembly K.E. E. T. EMBO J. 2003; PubMed Scopus Google Scholar, T. J. PubMed Scopus Google Scholar, N. M. R. T. Luhrmann R. EMBO J. PubMed Scopus Google Scholar). Finally, we note that the SPF30 and its interaction domains are not conserved in S. with there is no yeast homolog of does not have a similar central and human show in the C-terminal the C-terminal tails of SmD1, SmD3, and Lsm4 are not in yeast. The of these in but not in S. cerevisiae may be of the in splicing in these In S. cerevisiae have human that can be in and are The spliceosome assembly process in likely additional protein not found in yeast. is to that an of protein in higher to the of spliceosomes on transcripts. We and for critical on the and of the for and