Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry

Abstract

Recently, we identi®ed proteins that co-purify with the human spliceosome using mass spectrometry. One of the identi®ed proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5 + gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The ®rst catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The puri®ed CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identi®ed. This work reports a ®rst puri®cation and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least ®ve additional protein factors. Keywords: CDC5L/mass spectrometry/pre-mRNA splicing/spliceosome IntroductionNuclear pre-mRNA splicing is the process by which the introns (intervening sequences) in the primary transcript are speci®cally removed and the coding sequences joined to form mature mRNA, which is subsequently transported into the cytoplasm for protein synthesis. Splicing takes place in the nucleus, via a two-step transesteri®cation mechanism, and this is catalysed by a large RNA±protein complex termed the spliceosome. The major subunits of spliceosomes are the U1, U2, U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs), each of which contains the corresponding snRNA and a set of snRNP proteins (reviewed by Kra Èmer, 1996;Will and Lu Èrhmann, 1997). In addition, other non-snRNP protein splicing factors are also required for spliceosome formation and splicing (Will and Lu Èrhmann, 1997;Staley and Guthrie, 1998). Spliceosome assembly occurs in a pathway that involves the sequential binding of snRNP and protein splicing factors to conserved intron sequences to form the active complex. This is a multistep process and separate ATP-dependent complexes designated A, B and C have been identi®ed in vitro (reviewed by Reed and Palandjian, 1997). The C complex contains the active spliceosome while the A and B complexes contain assembly intermediates. The detailed roles played by many spliceosomal proteins in the pre-mRNA splicing mechanism are not yet fully understood.The protein composition of mammalian spliceosomes has been studied using complexes puri®ed from HeLa nuclear extracts by a combination of gel ®ltration and af®nity chromatography (Reed, 1990;Bennett et al., 1992). More recently in our laboratories, a large-scale analysis of spliceosome-associated proteins has been carried out using mass spectrometry and database searches (Neubauer et al., 1998). One...

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

“…1+ H-complex (this study)+ 2+ A/B-complex (Bennett et al+, 1992a)+ 3+ A/B-complex (Neubauer et al+, 1998)+ 4+ S. cerevisiae tri-snRNP (Gottschalk et al+, 1999)+ 5+ S. cerevisiae tri-snRNP (Stevens & Abelson, 1999)+ 6+ Human CDC5 complex (Ajuh et al+, 2000)+ 7+ C-complex (this study)+ ⅙ : proteins identified in this study by a single unique peptide only+…”

“…The complexes characterized by Bennett et al+ and Neubauer et al+ both contained a number of proteins involved in early spliceosome assembly, but not found in our C complex preparation+ These include some U1 snRNP proteins and first-step factors (see above)+ B and C complexes do share the core U2 and U5 snRNP proteins, as well as Prp19, SKIP, p68, CDC5, and PRL1+ However, a number of uncharacterized proteins found by Neubauer et al+ were not identified in C complex+ Several proteins shared between B and C complexes were also found associated with the tri-snRNP (U4/U6+U5) from Saccharomyces cerevisiae by mass spectrometry (Gottschalk et al+, 1999;Stevens & Abelson, 1999)+ These studies found the majority of Sm, LSM, and U5 snRNP proteins, as well as several new tri-snRNP proteins+ One of these, SART1, is present in C complex but was not identified in B complex+ The tri-snRNP associated proteins Prp3 and Prp4 are found in B complex, but Prp4 was identified by only a single peptide in C complex+ A U4/U6 associated protein, Prp4 has been shown to join the spliceosome as part of the tri-snRNP, but it appears to depart with U4 snRNA during the transition to C complex (Ayadi et al+, 1997)+ Also listed in Table 2 are proteins associated with the CDC5 protein, first identified as a novel spliceosome component in the mass spectrometry analysis of B complexes (Neubauer et al+, 1998)+ This protein has also been shown to associate with a 40S complex from Schizosaccharomyces pombe where it is essential for pre-mRNA splicing (Burns et al+, 1999;McDonald et al+, 1999)+ The 40S S. pombe complex also contains homologs to U5-220 (Prp8) and U5-116, as well as SmD2, Prp19, Prp5, Syf1, and Ecm2 (Slt11)+ Like C complex, the 40S S. pombe complex contains U2, U5, and U6 snRNAs, but no U1 or U4 (McDonald et al+, 1999)+ Mass spectrometry of immunopurified CDC5 complex from HeLa cells identified Sm proteins and U1 and U2 snRNP proteins, as well as a number of other splicingassociated proteins (Ajuh et al+, 2000)+ In the mammalian system, no U5 or tri-snRNP proteins were identified, although splicing intermediates were immunoprecipitated with CDC5+ Of the proteins associated with mammalian CDC5 that had not been previously linked to splicing, only one, HSP148, was also found in our C complex preparation+…”

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

“…Vectors for expression of GFP-Cdc5L and GFP-PLRG1 in mammalian cells were described previously [9]. hPrp19, Cdc5L, and Plrg1 cDNAs were subsequently cloned into the pEntr11 vector, and then moved to the pDest26, 27, 53 and pcDNA3.1/nV5 vectors of the Gateway cloning system (Invitrogen).…”

“…Based on epistasis analysis PSO4 was assigned to both the yeast RAD6 and RAD52 groups indicating the pleiotropic nature of this mutation [3,4]. Interestingly, cloning of PSO4 showed that it was allelic to PRP19 a previously characterized component of the pre-mRNA splicing complex in both yeast and human cells [5][6][7][8][9][10].…”

“…Based on epistasis analysis PSO4 was assigned to both the yeast RAD6 and RAD52 groups indicating the pleiotropic nature of this mutation [3,4]. Interestingly, cloning of PSO4 showed that it was allelic to PRP19 a previously characterized component of the pre-mRNA splicing complex in both yeast and human cells [5][6][7][8][9][10].The complete function of Prp19 in mRNA splicing is not known, however, it has been shown that the Prp19p-associated complex is required for activation of the pre-mRNA splicesome and for the stable association of the small nuclear RNAs U5 and U6 with the spliceosome after U4 is dissociated [11,12]. These findings suggest a possible structural role for the Prp19-associated complex in pre-mRNA splicing.…”