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- W2093485679 abstract "Two sequences important for pre-mRNA splicing precede the 3′ end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF65 to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF65 binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF65, arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF65binding. Two sequences important for pre-mRNA splicing precede the 3′ end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF65 to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF65 binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF65, arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF65binding. U2 small nuclear ribonucleoprotein particle small nuclear RNA polypyrimidine glutathione S-transferase branch point mammalian branch point binding protein m7G(5′)ppp(5′ heterogeneous nuclear RNP commitment complex 2 The expression of eukaryotic genes requires the accurate removal of intervening sequences (introns) and the concomitant fusion of the flanking exons via RNA splicing. The correct recognition of 5′ and 3′ splice sites occurs in the spliceosome, a large and dynamic macromolecular complex of small nuclear ribonucleoprotein particles (snRNPs)1 and non-snRNP proteins that assembles in a stepwise manner on the pre-mRNA (1Burge C. Tuschl T. Sharp P. Gesteland R. Cech T. Atkins J. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 525-560Google Scholar,2Krämer A. Annu. Rev. Biochem. 1996; 65: 367-409Crossref PubMed Scopus (617) Google Scholar). Five snRNPs play a role in removal of canonical GU/AG introns, each composed of a different U snRNA, a set of polypeptides common to most spliceosomal snRNPs and a set of proteins specific for each snRNP (reviewed in Refs. 3Will C. Behrens S. Lührmann R. Mol. Biol. Rep. 1993; 18: 121-126Crossref PubMed Scopus (37) Google Scholar and 4Kambach C. Walke S. Nagai K. Curr. Opin. Struct. Biol. 1999; 9: 222-230Crossref PubMed Scopus (110) Google Scholar).The first ATP-dependent step in spliceosome assembly is the stable association of U2 snRNP with the 3′ part of the intron (reviewed in Refs. 5Hodges P. Beggs J. Curr. Biol. 1994; 4: 264-267Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar and 6Reed R. Curr. Opin. Genet. & Dev. 1996; 6: 215-220Crossref PubMed Scopus (247) Google Scholar). This part of the pre-mRNA contains three sequence elements important for the splicing process as follows: the branch point region (BP), the polypyrimidine tract (Py tract), and the conserved dinucleotide AG at the 3′ splice site. The BP is highly conserved in yeast (UACUAAC) and is more degenerate in higher eukaryotes (consensus YNCURAY, Y is pyrimidine, R is purine, N is any nucleotide). The BP establishes base pairing interactions with a specific sequence of U2 snRNA, bulging out the nucleotide, usually adenosine, that forms a 2′–5′ phosphodiester bond with the 5′ end of the intron (7Parker R. Siliciano P. Guthrie C. Cell. 1987; 49: 229-239Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 8Wu J. Manley J. Genes Dev. 1989; 3: 1553-1561Crossref PubMed Scopus (211) Google Scholar, 9Zhuang Y. Weiner A. Genes Dev. 1989; 3: 1545-1552Crossref PubMed Scopus (201) Google Scholar, 10Nelson K. Green M. Genes Dev. 1989; 3: 1562-1571Crossref PubMed Scopus (71) Google Scholar). The Py tract, particularly important for splicing in higher eukaryotes, is a pyrimidine-rich sequence located immediately downstream of the BP and upstream of the AG dinucleotide (1Burge C. Tuschl T. Sharp P. Gesteland R. Cech T. Atkins J. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 525-560Google Scholar).Biochemical fractionation of mammalian nuclear extracts indicated that stable binding of purified U2 snRNP to the pre-mRNA requires four activities as follows: SF3a, SF3b, U2AF, and SF1 (11Krämer A. Utans U. EMBO J. 1991; 10: 1503-1509Crossref PubMed Scopus (68) Google Scholar). SF3a and SF3b were subsequently found to be integral components of the 17 S U2 snRNP that dissociate during purification (12Behrens S. Tyc K. Kastner B. Reichelt J. Lührmann R. Mol. Cell. Biol. 1993; 13: 307-319Crossref PubMed Scopus (98) Google Scholar). U2AF is composed of two subunits of 65 and 35 kDa (13Zamore P. Green M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9243-9247Crossref PubMed Scopus (277) Google Scholar). U2AF65 binds to the Py tract (14Zamore P. Patton J. Green M. Nature. 1992; 355: 609-614Crossref PubMed Scopus (457) Google Scholar), whereas U2AF35 recognizes the 3′ splice site AG and helps to stabilize the U2AF65/Py tract interaction (15Wu S. Romfo C. Nilsen T. Green M. Nature. 1999; 402: 832-835Crossref PubMed Scopus (239) Google Scholar, 16Zorio D. Blumenthal T. Nature. 1999; 402: 835-838Crossref PubMed Scopus (190) Google Scholar, 17Merendino L. Guth S. Bilbao D. Martinez C. Valcárcel J. Nature. 1999; 402: 838-841Crossref PubMed Scopus (219) Google Scholar). SF1, also called mammalian branch point binding protein (mBBP), was purified as a 75-kDa polypeptide and found to recognize specifically the BP (18Krämer A. Mol. Cell. Biol. 1992; 12: 4545-4552Crossref PubMed Scopus (72) Google Scholar, 19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).The mechanisms by which U2AF and SF1/mBBP facilitate U2 snRNP binding are not well understood. The amino-terminal arginine-serine (RS) rich domain of U2AF65 contacts the BP, and it has been proposed that its positively charged residues can stabilize the limited base pairing interactions that can be established between U2 snRNA and the BP (20Lee C. Zamore P. Green M. Hurwitz J. J. Biol. Chem. 1993; 268: 13472-13478Abstract Full Text PDF PubMed Google Scholar, 21Gaur R. Valcárcel J. Green M. RNA (NY ). 1995; 1: 407-417PubMed Google Scholar, 22Valcárcel J. Gaur R. Singh R. Green M. Science. 1996; 273: 1706-1709Crossref PubMed Scopus (226) Google Scholar). An interaction between U2AF65 and SAP 155, a protein subunit of SF3b, can provide an additional mechanism for U2 recruitment (23Gozani O. Potashkin J. Reed R. Mol. Cell. Biol. 1998; 18: 4752-4760Crossref PubMed Scopus (225) Google Scholar). U2AF65 also interacts with UAP56, a DEAH box helicase found to be important for U2 snRNP binding (24Fleckner J. Zhang M. Valcárcel J. Green M. Genes Dev. 1997; 11: 1864-1872Crossref PubMed Scopus (209) Google Scholar).SF1/mBBP and U2AF65 interact with each other, and this interaction can facilitate cooperative recognition of the BP and Py tract that are adjacent to each other in the pre-mRNA (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 26Berglund J. Abovich N. Rosbash M. Genes Dev. 1998; 12: 858-867Crossref PubMed Scopus (189) Google Scholar). It is currently unclear whether SF1/mBBP plays other roles in promoting U2 snRNP assembly.SF1/mBBP contains at least five distinct structural domains. The amino-terminal region contacts U2AF65 (27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar), an essential interaction for SF1/mBBP function that is disrupted by phosphorylation by the serine/threonine kinase PKG-1 in response to cGMP (28Wang X. Bruderer S. Rafi Z. Xue J. Milburn P. Krämer A. Robinson P. EMBO J. 1999; 18: 4549-4559Crossref PubMed Scopus (75) Google Scholar). This region of the protein is followed by an hnRNP K homology domain (KH domain) and a zinc knuckle, two motifs implicated in RNA binding (reviewed in Ref. 29Nagai K. Curr. Opin. Struct. Biol. 1996; 6: 53-61Crossref PubMed Scopus (159) Google Scholar). The KH domain is sufficient for specific recognition of the pre-mRNA BP (27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar, 30Berglund J. Fleming M. Rosbash M. RNA (NY ). 1998; 4: 998-1006Crossref PubMed Scopus (39) Google Scholar), and the zinc knuckle seems to confer additional RNA binding affinity, probably through interaction with the negatively charged phosphate backbone (30Berglund J. Fleming M. Rosbash M. RNA (NY ). 1998; 4: 998-1006Crossref PubMed Scopus (39) Google Scholar). The RNA binding domains are followed by a proline-rich region and a carboxyl-terminal domain. Alternatively spliced variants of human SF1/mBBP are expressed in a cell type-specific manner that differ in the length of the proline-rich region and have distinct carboxyl termini (31Arning S. Grüter P. Bilbe G. Krämer A. RNA (NY ). 1996; 2: 794-810PubMed Google Scholar, 32Krämer A. Quentin M. Mulhauser F. Gene (Amst. ). 1998; 211: 29-37Crossref PubMed Scopus (14) Google Scholar). The carboxyl terminus and the proline-rich region are not only dispensable for SF1/mBBP activity in spliceosome assemblyin vitro but also for viability in yeast (27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar) and might be required for other functions of SF1/mBBP in vivo.A Saccharomyces cerevisiae SF1/mBBP homolog was genetically identified by its synthetic lethality with MUD2, the gene encoding the functional homolog of U2AF65 in yeast (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Biochemical experiments showed that ySF1/BBP also contacts the 3rd RNA Recognition Motif of Mud2p and binds the BP (19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Direct interaction of ySF1/BBP with prp40, a component of U1 snRNP, suggests that ySF1/BBP can form a bridge between the 5′ splice site and the 3′ splice site region in one of the first detectable complexes able to commit the pre-mRNA to undergo splicing (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar). Human SF1/mBBP has also been found to be a component of such complexes in mammals (19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar,25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). SF1/mBBP is not present, however, in pre-spliceosome complexes, suggesting that BP recognition by the protein is no longer needed and/or is incompatible with the stable association of U2 snRNP with this sequence (19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar). Recent results suggest that ySF1/BBP plays a kinetic role in the progression through the two commitment complexes observable in yeast extracts (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar). Depletion of ySF1/BBP resulted in a reduction in the accumulation of commitment complex 2 (CC2), in which the 5′ splice site is recognized by U1 snRNP and the BP by ySF1/BBP and MUD2. Surprisingly, however, spliceosome formation was not affected by ySF1/BBP depletion. These results suggest that under conditions of reduced amounts of ySF1/BBP, formation of CC2 became rate-limiting and the CC2 complexes formed were immediately chased into spliceosomes (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar).In this report we have developed an immunodepletion protocol allowing removal of SF1/mBBP from HeLa nuclear extracts, and we used this method to answer two questions. First, is the activity of SF1/mBBP essential for U2 snRNP binding in mammals but more dispensable in yeast, as suggested by the differences between biochemical reconstitution experiments in HeLa extracts and depletion experiments in S. cerevisiae? Second, is the only role of SF1/mBBP in mammalian spliceosome assembly to assist U2AF65 binding to the Py tract? Consistent with the results in yeast and in contrast with the requirement of SF1/mBBP in reconstituted reactions with purified components, extensive depletion of SF1 had relatively small effects in the accumulation of complexes containing U2 snRNP. Although substitution of the weak BP of an IgM pre-mRNA by the yeast BP consensus conferred a kinetic advantage in spliceosome assembly, this advantage was no longer observed in SF1-depleted nuclear extracts. These effects correlated with a reduced cross-link of U2AF65 to the RNA and could be reversed by supplementing the depleted extract with recombinant SF1/mBBP. Interestingly, addition of U2AF65 also increased the cross-linking signal but was unable to restore splicing complex formation. These observations imply additional functions for SF1/mBBP in spliceosome assembly distinct from facilitating U2AF65 binding.DISCUSSIONSF1/mBBP was first identified as a biochemical activity necessary, together with U2AF, to allow binding of purified human U2 snRNP to a model pre-mRNA (11Krämer A. Utans U. EMBO J. 1991; 10: 1503-1509Crossref PubMed Scopus (68) Google Scholar). SF1/mBBP was subsequently found to be essential for the viability of S. cerevisiae andCaenorhabditis elegans (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 42Mazroui R. Puoti A. Krämer A. RNA (NY ). 1999; 5: 1615-1631Crossref PubMed Scopus (37) Google Scholar). The essential nature of theSF1/mBBP gene could be due to the activity of its protein products in pre-mRNA splicing. Depletion experiments in yeast (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar) and in mammalian extracts (Figs. 2 and 3), however, indicate that significant reduction of SF1/mBBP levels cause only modest changes in the efficiency of formation of splicing complexes and in the accumulation of splicing products. It is possible that the splicing defects are more pronounced for pre-mRNAs of some essential gene(s) than for the model pre-mRNAs used in these studies. Indeed, the results of Figs. 2 and 3 are consistent with the possibility that the effects of SF1/mBBP depletion can be quantitatively different for different pre-mRNA substrates. Splicing defects upon ySF1/BBP depletion were also more readily noticeable in S. cerevisiaewhen reporters with mutated splice sites were analyzed. 2B. Rutz and B. Séraphin, personal communication. Alternatively, the essential function(s) of SF1/BBP may be unrelated to pre-mRNA splicing. In line with the latter possibility, one SF1/mBBP isoform, ZFM1, was independently identified as a transcriptional repressor (43Zhang D. Paley A. Childs G. J. Biol. Chem. 1998; 273: 18086-18091Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).The low levels of ySF1/BBP required for viability and splicing of model substrates are particularly striking considering that S. cerevisiae cells in which transcription of the ySF1/BBPgene has been efficiently turned off can live for several generations with only some delay in their doubling time (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar). Rutz and Séraphin (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar) have interpreted their observations as revealing a transient interaction of ySF1/BBP with splicing complexes that kinetically facilitates their assembly. A similar transient interaction was also observed for MUD2p, the yeast homolog of U2AF65. As yeast SF1/BBP and MUD2p interact with each other through protein domains conserved in their mammalian counterparts (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar), both proteins may bind to and leave the pre-mRNA as a complex, at least in the yeast system.Depletion of mammalian SF1/mBBP and of U2AF65, however, have dramatically different effects in spliceosome assembly. The relatively modest effects of SF1/BBP depletion are in contrast with the substantial loss of activity of extracts chromatographically depleted or immunodepleted of U2AF65 (14Zamore P. Patton J. Green M. Nature. 1992; 355: 609-614Crossref PubMed Scopus (457) Google Scholar, 21Gaur R. Valcárcel J. Green M. RNA (NY ). 1995; 1: 407-417PubMed Google Scholar, 22Valcárcel J. Gaur R. Singh R. Green M. Science. 1996; 273: 1706-1709Crossref PubMed Scopus (226) Google Scholar, 24Fleckner J. Zhang M. Valcárcel J. Green M. Genes Dev. 1997; 11: 1864-1872Crossref PubMed Scopus (209) Google Scholar, 36Guth S. Martinez C. Gaur R. Valcárcel J. Mol. Cell. Biol. 1999; 19: 8263-8271Crossref PubMed Scopus (73) Google Scholar, 47Gama-Carvalho M. Krauss R.D. Chiang L. Valcárcel J. Green M.R. Carmo-Fonseca M. J. Cell Biol. 1997; 137: 975-987Crossref PubMed Scopus (110) Google Scholar). This is despite the fact that the two proteins bind cooperatively to the same region of higher eukaryotic pre-mRNAs, act at a similar step in spliceosome formation, their levels in HeLa nuclear extracts are comparable, and the extent of the reduction of their concentration in the depleted extracts is also comparable (compare, for example, Fig. 3B in Ref. 36Guth S. Martinez C. Gaur R. Valcárcel J. Mol. Cell. Biol. 1999; 19: 8263-8271Crossref PubMed Scopus (73) Google Scholar with Fig. 1 in this work). The dissociation constants for SF1/BBP binding to the yeast consensus BP range between 0.5 (yeast) and 30 μm (human). The affinity of U2AF65 for typical polypyrimidine tracts is significantly higher, the correspondingly lower dissociation constants ranging between 0.1 and 0.01 μm. If the effect of the depletion would depend solely on the relative affinities of these factors for their cognate sites, one would expect that reduction in the levels of the factor that has higher binding affinity for its binding site would be less detrimental for activity. However, the opposite is observed. These observations may imply a more critical function for U2AF65than for SF1/mBBP in mammalian spliceosome assembly. Alternatively, they may reflect additional roles that U2AF65 plays in spliceosome assembly and that are independent of SF1/mBBP. In any case, the results argue against the possibility that the recognition of the more degenerate mammalian BP requires a more substantial contribution of SF1/mBBP compared with that of the conserved branch point by ySF1/BBP in the yeast system.The strong dependence on SF1/mBBP to reconstitute U2 snRNP binding from purified components (11Krämer A. Utans U. EMBO J. 1991; 10: 1503-1509Crossref PubMed Scopus (68) Google Scholar) is in contrast with the modest effects that SF1/BBP depletion has in the same process in extracts (Figs. Figure 2, Figure 3, Figure 4). It is possible that other factors present in the nuclear extract can mitigate the effects of SF1/mBBP depletion by either facilitating alternative pathways of assembly or by taking over SF1/mBBP function in the immunodepleted extracts.A key and still unsolved question is the molecular mechanism(s) by which SF1/BBP promotes U2 snRNP binding and/or other events in spliceosome assembly. SF1/BBP was proposed to be involved in initial cross-intron bridging by interacting simultaneously with the branch point sequence and Prp40p, which is associated with U1snRNP bound to the 5′ splice site (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Both synthetic lethality in yeast and GST-pulldown experiments were compatible with this proposal (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). The proline-rich region of yeast SF1/BBP and the WW domain of PrP40p were suggested to mediate this interaction. Analogous interactions were proposed in the mammalian system, involving the WW domain of the formin-binding protein FBP21 that can interact with human SF1/BBP, the U1-specific polypeptide U1C, and the snRNP core components SmB/SmB′ (44Bedford M. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Crossref PubMed Scopus (134) Google Scholar). Several results, however, have challenged the role of these interactions in the initial bridging between splice sites. First, domain mapping analysis showed that the proline-rich region of ySF1/BBP located in the carboxyl-terminal half of the protein was not required for binding to Prp40p (25Abovich N. Rosbash M. Cell. 1997; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Second, FBP21 has been found in pre-spliceosome and spliceosome (A and B) complexes, probably associated to U2 snRNP, but not in commitment (E) complexes (44Bedford M. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Crossref PubMed Scopus (134) Google Scholar). Third, the proline-rich region was found to be dispensable for promoting mammalian U2 snRNP binding in reconstituted reactions from purified components (27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar) and in SF1/mBBP-depleted extracts (Fig. 2) and also for yeast growth (27Rain J. Rafi Z. Rhani Z. Legrain P. Krämer A. RNA (NY ). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar).Given that SF1/mBBP recognizes the nucleotide bases at the BP and that these bases also establish base pairing interactions with U2 snRNA, it seems unlikely that SF1/mBBP and U2 snRNA can be bound simultaneously at the BP sequence. In fact, previous data are consistent with SF1/mBBP being replaced from the BP upon U2 snRNP binding (33Rutz B. Séraphin B. RNA (NY ). 1999; 5: 819-831Crossref PubMed Scopus (76) Google Scholar, 45MacMillan A. Query C. Allerson C. Chen S. Verdine G. Sharp P. Genes Dev. 1994; 8: 3008-3020Crossref PubMed Scopus (131) Google Scholar, 46Chiara M. Gozani O. Bennett M. Champion-Arnaud P. Palandjian L. Reed R. Mol. Cell. Biol. 1996; 16: 3317-3326Crossref PubMed Scopus (94) Google Scholar). SF1/mBBP could interact with a protein component of U2 snRNP or with U2 snRNA to bring the snRNP in close proximity to the BP, or to induce conformational rearrangements within the snRNP necessary for its recruitment, and then be displaced when extensive RNA-RNA and RNA-protein interactions stabilize snRNP binding to the BP. SF1/mBBP could act in this case as a “guide” molecule that identifies the correct sequence for U2 snRNP binding, a task particularly demanding in higher eukaryotes where the BP sequence is rather degenerate and several base pairing arrangements between U2 snRNA and the BP region are possible. This activity could be at the basis of the improved kinetics of U2 snRNP binding afforded by an efficient BP-SF1/BBP interaction observed in the experiments presented in Fig. 3.Alternatively, SF1/mBBP could help (with the assistance of U2AF65) to clear the BP region from factors associated with the pre-mRNA before its commitment to spliceosome assembly, like hnRNP proteins. In fact, Chiara et al. (46Chiara M. Gozani O. Bennett M. Champion-Arnaud P. Palandjian L. Reed R. Mol. Cell. Biol. 1996; 16: 3317-3326Crossref PubMed Scopus (94) Google Scholar) have shown that hnRNPI/PTB can be cross-linked to the BP sequence of a model pre-mRNA in those complexes. These factors may have more relaxed binding specificity but represent, because of their associated domains, activities or factors, a significant block to U2 snRNP binding and therefore their substitution by SF1/BBP could result in effective clearance of the BP region for U2 snRNP binding, particularly in combination with the cooperative binding and recruiting activities of U2AF65.The interaction between SF1/BBP and U2AF65 mutually facilitates their interaction with the BP and Py tract (19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). One possible scenario is that SF1 simply assists U2AF65 binding to the Py tract and that only U2AF65 has a recruitment function for U2 snRNP. RNA binding experiments using purified proteins have shown that SF1/BBP increases the affinity of U2AF655-fold (19Berglund J. Chua K. Abovich N. Reed R. Rosbash M. Cell. 1997; 89: 781-787Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). This increase in affinity may be higher for pre-mRNAs containing weak Py tracts and more consensus BPs. The results of Fig.4, however, argue that the function of SF1/BBP cannot be justified exclusively by its effects on U2AF65 binding, and argue that additional mechanisms like those discussed above can indeed play a role in SF1/BBP function. It is also possible that the interaction of SF1/BBP with U2AF65 enhances the recruitment activities of the latter, for example by triggering a conformational change that will allow more extensive contacts between U2AF65 and U2 snRNP components like SAP 155 (23Gozani O. Potashkin J. Reed R. Mol. Cell. Biol. 1998; 18: 4752-4760Crossref PubMed Scopus (225) Google Scholar). The expression of eukaryotic genes requires the accurate removal of intervening sequences (introns) and the concomitant fusion of the flanking exons via RNA splicing. The correct recognition of 5′ and 3′ splice sites occurs in the spliceosome, a large and dynamic macromolecular complex of small nuclear ribonucleoprotein particles (snRNPs)1 and non-snRNP proteins that assembles in a stepwise manner on the pre-mRNA (1Burge C. Tuschl T. Sharp P. Gesteland R. Cech T. Atkins J. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 525-560Google Scholar,2Krämer A. Annu. Rev. Biochem. 1996; 65: 367-409Crossref PubMed Scopus (617) Google Scholar). Five snRNPs play a role in removal of canonical GU/AG introns, each composed of a different U snRNA, a set of polypeptides common to most spliceosomal snRNPs and a set of proteins specific for each snRNP (reviewed in Refs. 3Will C. Behrens S. Lührmann R. Mol. Biol. Rep. 1993; 18: 121-126Crossref PubMed Scopus (37) Google Scholar and 4Kambach C. Walke S. Nagai K. Curr. Opin. Struct. Biol. 1999; 9: 222-230Crossref PubMed Scopus (110) Google Scholar). The first ATP-dependent step in spliceosome assembly is the stable association of U2 snRNP with the 3′ part of the intron (reviewed in Refs. 5Hodges P. Beggs J. Curr. Biol. 1994; 4: 264-267Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar and 6Reed R. Curr. Opin. Genet. & Dev. 1996; 6: 215-220Crossref PubMed Scopus (247) Google Scholar). This part of the pre-mRNA contains three sequence elements important for the splicing process as follows: the branch point region (BP), the polypyrimidine tract (Py tract), and the conserved dinucleotide AG at the 3′ splice site. The BP is highly conserved in yeast (UACUAAC) and is more degenerate in higher eukaryotes (consensus YNCURAY, Y is pyrimidine, R is purine, N is any nucleotide). The BP establishes base pairing interactions with a specific sequence of U2 snRNA, bulging out the nucleotide, usually adenosine, that forms a 2′–5′ phosphodiester bond with the 5′ end of the intron (7Parker R. Siliciano P. Guthrie C. Cell. 1987; 49: 229-239Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 8Wu J. Manley J. Genes Dev. 1989; 3: 1553-1561Crossref PubMed Scopus (211) Google Scholar, 9Zhuang Y. Weiner A. Genes Dev. 1989; 3: 1545-1552Crossref PubMed Scopus (201" @default.
- W2093485679 created "2016-06-24" @default.
- W2093485679 creator A5022884999 @default.
- W2093485679 creator A5039897914 @default.
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