Drosophila MFAP1 Is Required for Pre-mRNA Processing and G2/M Progression
2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês
10.1074/jbc.m803512200
ISSN1083-351X
AutoresDitte S. Andersen, Nicolas Tapon,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions (Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component (Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938-2946), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G2/M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G2/M arrest might be a general consequence of interfering with spliceosome function. The mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions (Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component (Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938-2946), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G2/M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G2/M arrest might be a general consequence of interfering with spliceosome function. Splicing of pre-mRNAs is a catalytic reaction that involves two successive trans-esterification steps. This process is carried out by a highly conserved ribonucleoprotein complex, called the spliceosome. The spliceosome consists of five snRNAs (U1, U2, U4, U5, and U6) and more than 200 proteins, making it one of the largest and most complex molecular machines studied (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Spliceosome assembly is a sequential process that is initiated by the recruitment of the U1 small nuclear ribonucleoprotein (snRNP) 2The abbreviations used are: snRNP, small nuclear ribonucleoprotein; qPCR, quantitative PCRs; MFAP1, microfibrillar-associated protein 1; NTc, 19 complex; RNAi, RNA interference; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; dsRNA, double-stranded RNA; BrdUrd, bromodeoxyuridine; RT, reverse transcriptase; GFP, green fluorescent protein; PH3, anti-phospho-histone 3. to the 5′-splice donor site to form a "commitment complex" ("E complex" in mammals) (3Rosbash M. Seraphin B. Trends Biochem. Sci. 1991; 16: 187-190Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 4Ruby S.W. Abelson J. Science. 1988; 242: 1028-1035Crossref PubMed Scopus (163) Google Scholar). Subsequently, recruitment of the U2 snRNP to the branch site by the U2 auxiliary factor of 65 kDa (U2AF65) generates the "pre-spliceosome" ("A complex" in mammals) (5Zorio D.A. Blumenthal T. Nature. 1999; 402: 835-838Crossref PubMed Scopus (193) Google Scholar, 6Wu S. Romfo C.M. Nilsen T.W. Green M.R. Nature. 1999; 402: 832-835Crossref PubMed Scopus (242) Google Scholar, 7Merendino L. Guth S. Bilbao D. Martinez C. Valcarcel J. Nature. 1999; 402: 838-841Crossref PubMed Scopus (223) Google Scholar, 8Singh R. Banerjee H. Green M.R. RNA (Cold Spring Harbor). 2000; 6: 901-911Google Scholar). A preformed U4/U6.U5 tri-snRNP unit then joins the U1-U2-pre-mRNA complex to form the "complete spliceosome" ("B complex" in mammals). To activate the complete spliceosome several conformational rearrangements must take place (reviewed in Ref. 9Brow D.A. Annu. Rev. Genet. 2002; 36: 333-360Crossref PubMed Scopus (295) Google Scholar). These include unwinding of base pairings between U1 and the 5′-splice site and between U6 and U4, as well as the formation of a new base pair interaction between U5 and U6 snRNPs and the 5′-splice site and U6 and U2. As a result, U1 and U4 snRNPs are released and an active spliceosome is formed (10Madhani H.D. Guthrie C. Annu. Rev. Genet. 1994; 28: 1-26Crossref PubMed Scopus (319) Google Scholar, 11Staley J.P. Guthrie C. Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). The yeast RNA helicases Prp28p and Brr2p are both implicated in the structural rearrangements that take place during the activation step of the spliceosome (11Staley J.P. Guthrie C. Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). Prp28p is thought to be important for the unwinding of base pairings between U1 and the 5′-splice site (12Chen J.Y. Stands L. Staley J.P. Jackups Jr., R.R. Latus L.J. Chang T.H. Mol. Cell. 2001; 7: 227-232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and Brr2p is implicated in unwinding the U4/U6 duplex, which is essential for the release of U4 snRNP (13Raghunathan P.L. Guthrie C. Curr. Biol. 1998; 8: 847-855Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Moreover, a pre-assembled Prp19p (pre-mRNA processing factor 19 protein)-associated protein complex named the 19 complex (NTc) in yeast and the Cdc5-Prp19 complex in humans is required for maturation of the spliceosome (14Chen C.H. Tsai W.Y. Chen H.R. Wang C.H. Cheng S.C. J. Biol. Chem. 2001; 276: 488-494Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (153) Google Scholar). Much of the mechanistic insight into the spliceosome has been derived from yeast genetics. The ease of generating temperature-sensitive mutant alleles has allowed a detailed functional characterization of individual splicing factors. In contrast, the function of the mammalian spliceosome has mostly been studied by proteomic analysis of spliceosome intermediates (15Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (153) Google Scholar, 16Bessonov S. Anokhina M. Will C.L. Urlaub H. Luhrmann R. Nature. 2008; 452: 846-850Crossref PubMed Scopus (299) Google Scholar, 17Chen Y.I. Maika S.D. Stevens S.W. J. Mol. Biol. 2006; 361: 412-419Crossref PubMed Scopus (7) Google Scholar, 18Deckert 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, 19Jurica M.S. Licklider L.J. Gygi S.R. Grigorieff N. Moore M.J. RNA (Cold Spring Harbor). 2002; 8: 426-439Google Scholar, 20Makarov E.M. Makarova O.V. Urlaub H. Gentzel M. Will C.L. Wilm M. Luhrmann R. Science. 2002; 298: 2205-2208Crossref PubMed Scopus (305) Google Scholar, 21Neubauer 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 (421) Google Scholar). The isolation and comparison of different spliceosome intermediates has given insights into the dynamics of the spliceosome and revealed that excision of an intron from a pre-mRNA occurs largely by the same sequential path in yeast and higher eukaryotes (15Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (153) Google Scholar, 16Bessonov S. Anokhina M. Will C.L. Urlaub H. Luhrmann R. Nature. 2008; 452: 846-850Crossref PubMed Scopus (299) Google Scholar, 19Jurica M.S. Licklider L.J. Gygi S.R. Grigorieff N. Moore M.J. RNA (Cold Spring Harbor). 2002; 8: 426-439Google Scholar, 20Makarov E.M. Makarova O.V. Urlaub H. Gentzel M. Will C.L. Wilm M. Luhrmann R. Science. 2002; 298: 2205-2208Crossref PubMed Scopus (305) Google Scholar). Importantly, purifications of mammalian spliceosomes have added a vast array of elements to the pre-existing list of conserved core splicing factors. Many of these have no previous connection to splicing and require functional validation (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Yeast Prp38p is a U4/U6.U5 tri-snRNP component that plays an important role in the maturation of the spliceosome. Prp38p activity is dispensable for the initial assembly of the spliceosome, but is required in a later step for the release of U4 snRNP and activation of the spliceosome (2Xie J. Beickman K. Otte E. Rymond B.C. EMBO J. 1998; 17: 2938-2946Crossref PubMed Scopus (48) Google Scholar). It has been speculated that Prp38p might recruit (or activate) an RNA unwinding activity necessary for the release of U4 snRNA and the integration of U6 snRNA into the active site of the spliceosome (2Xie J. Beickman K. Otte E. Rymond B.C. EMBO J. 1998; 17: 2938-2946Crossref PubMed Scopus (48) Google Scholar). The identity of this RNA unwinding activity remains unknown, but Brr2p is a possible candidate. The mammalian Prp38p homologue does not appear to be stably associated with the tri-snRNPs and has been recovered in surprisingly few spliceosomal purifications (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). This apparent discrepancy between yeast and higher eukaryotes prompted us to study the Drosophila Prp38 protein in more detail. We initially used a proteomics approach to identify proteins associated with dPrp38. In agreement with its function in yeast, dPrp38 associates with several homologues of splicing factors that are required for activation of the spliceosome. In addition, we identified dMFAP1 (microfibrillar-associated protein 1), which binds directly to dPrp38. We show that dMFAP1, like dPrp38, associates with spliceosome components and is required for pre-mRNA processing. Finally, we find that dPrp38 and dMFAP1 are required for normal string/cdc25 mRNA levels and G2/M progression in cultured cells and for cell survival and growth in vivo. Generation of dprp38 Mutant and Transgenic Flies—The G19491 P-element (Genexel Inc.) was inserted 798 bp into the open reading frame of dprp38 (dprp38G19491). The dprp38E1 mutant was generated by mobilization of the G19491 P-element using standard genetic techniques and identified by PCR using the following primers: dPrp38-5.03, CTTTGCTCTCACTGCGGAGCT; dPrp38-3.3: AGCGAGCAAGTAGGAACGGAACGGAACGGC. In dprp38E1, the region between bp 428 and 798 of the open reading frame has been deleted. The dmfap1 D2-1 RNAi line was generated by cloning two 400-bp inverted repeats of dmfap1 into the pMF3 vector using the unique EcoRI and XbaI restriction sites. The 400-bp repeats were amplified from genomic DNA using a forward primer containing a BglII site at the 5′ end and reverse primers containing an EcoRI or a XbaI site at the 3′ end: dMFAP1ForwBglII, GAGAGATCTCGACGAGGTGGAATACGAGG; dMFAP1RevEco, GGAATTCCGAACTTGGTGGTGTCCTGG; dMFAP1RevXba, GTCTAGACGAACTTGGTGGTGTCCTGG. The two PCR products were digested with BglII and EcoRI or BglII and XbaI and cloned into the same pMF3 vector digested with EcoRI and XbaI. The final construct was introduced into the germline by injections in the presence of transposase as previously described (22Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2340) Google Scholar, 23Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar). The dMFAP1 (15610) and dPrp38 (21136) RNAi lines were obtained from the Vienna Drosophila RNAi Center. Genotypes—The following genotypes were used: wiso (Fig. 1, C and D). hs-FLP; FRT42D, dprp38G19491/FRT42D Ubi-GFP (Fig. 1, F and G). hs-FLP; FRT42D, Ubi-GFP, dprp38E1/FRT42D, M(2)531 (Fig. 1, H-K). hs-FLP; Act >cd2 > Gal4, UAS-GFP (Fig. 5, A-C). hs-FLP; UAS-RNAi-dmfap1/Act > cd2 > Gal4, UAS-GFP (Fig. 5, D-F). hs-FLP; UAS-RNAi-dprp38/Act > cd2 > Gal4, UAS-GFP (Fig. 5, G-I).FIGURE 5dPrp38 and dMFAP1 are required for G2/M progression and developmental growth and proliferation. A-I, wing imaginal dics from third instar larvae. Posterior is to the right. Wild-type clones (A-C) or clones expressing dprp38 and dmfap1 RNAi constructs (D-I) were generated using the FLP-out technique (marked with GFP in A, D, and G). Clones with reduced levels of dMFAP1 (D-F) or dPrp38 (G-I) are smaller than control clones (A-C) generated at the same time and undergo apoptosis as measured by increased levels of cleaved caspase 3 staining in the clones (red in E and H). J-K, cells depleted of dMFAP1 arrest in G2/M. S2 cells were treated with dsRNA targeting eGFP (control) or dmfap1 for 3 days. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The ratio of cells in the G2/M relative to G1 phase is indicated for each treatment (G1:G2/M). L, immunoblotting confirms that dMFAP1 protein levels are reduced in cells treated with dsRNA targeting dmfap1 compared with control cells. Anti-β-tubulin is used as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunohistochemistry—Mosaic tissues were obtained using the hs-Flp/FRT system (24Xu T. Rubin G.M. Development. 1993; 117: 1223-1237Crossref PubMed Google Scholar). Salivary glands and wing and eye imaginal discs were dissected from L3 larvae (120 h after egg laying) in 1× PBS. S2 cells were seeded at a density of 5 × 106/ml on chamber slides (Nunc, Fig. 6, J-M). Tissues and cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature, washed four times in PBS containing 0.1% Triton X-100 (PBS-T), blocked for 2 h in PBS-T containing 10% goat serum (PBS-TG), and incubated with primary antibodies in PBS-TG overnight at 4 °C. Rabbit anti-cleaved caspase-3 (Asp175) (Cell Signaling), rabbit anti-dPrp38_C, mouse anti-α-tubulin (Sigma), and rabbit anti-phospho-histone H3 (PH3) (Upstate) were used at 1:500. The next day, cells and tissues were washed, blocked in PBS-TG, and incubated with secondary antibodies at 1:500 (rhodamine red X donkey anti-rabbit, anti-mouse, anti-guinea pig, and fluorescein (isothiocyanate) donkey anti-rabbit from Jackson ImmunoResearch) for 2 h at room temperature. Hoechst (Sigma) was added to the secondary antibody mixture during the last 30 min of the incubation to stain DNA (Fig. 1D). After washes, cells and tissues were mounted in Vectashield. Fluorescence images were acquired using a Zeiss LSM510 Confocal Laser Scanning Microscope (×25 and 40 objectives) and processed using Adobe photoshop CS2. Cell Culture—Drosophila S2 cells were grown in Schneider's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen) at 25 °C. Transfections were done using Effectene (Qiagen). Antibodies—The anti-dPrp38_N and anti-dPrp38_C rabbit antibodies and the anti-dMFAP1 guinea pig antibody were generated and affinity purified by Eurogentec SA (Seraing, Belgium) against peptides corresponding to amino acids 1-15 and 315-330 of dPrp38 and 43-57 of dMFAP1. Plasmids—dPrp38Δ, dPrp38G19491, dMFAP1ΔN, and dMFAP1ΔC were PCR amplified and cloned into the pENTR™/D-TOPO vector using gene-specific primers: for dPrp38Δ, sense primer, CACCATGGCCAACCGCACGGTGAAGG, antisense primer, GATTTCGTTGTTTTCCTCGAG; for dPrp38G19491, sense primer, CACCATGGCCAACCGCACGGTGAAGG, antisense primer, TCCTCGTTGTGGGTGTCCCG; for dMFAP1ΔN, sense primer, CACCATGGACAACGAACCCCGCCTGAAG, antisense primer, TTACTCCATCTTTTTTCGCTTCG; and for dMFAP1ΔC, sense primer, CACCATGAGTGCAGCCACCGCCGCCG, antisense primer, CTCCTCGCTTTCGGTCTCCTC. To generate HA-dPrp38Δ, HA-dPrp38G19491, HA-dMFAP1ΔN, and HA-dMFAP1ΔC, dPrp38Δ, dPrp38G19491, dMFAP1ΔN, and dMFAP1ΔC were cloned into the Gateway pAHW vector (Drosophila Gateway Vector Collection). To generate GST-dMFAP1, dMFAP1 was PCR amplified using gene-specific sense and antisense primers containing EcoRI and NotI restriction sites, respectively, and subcloned into the pGEX4T-1 vector (Amersham Biosciences) using the EcoRI and NotI restriction sites. To generate His-dPrp38, dPrp38 was PCR amplified using gene-specific sense and antisense primers containing KpnI and HindIII restriction sites, respectively, and subcloned into the pRSET-A vector (Invitrogen) using KpnI and HindIII restriction sites. Immunoprecipitation and GST Pull-down—Immunoprecipitations of dPrp38 and dMFAP1 were performed from 1 × 109 (Figs. 3B and 4B) or 1 × 107 (Fig. 3, D and G-J) S2 cells. Cells were lysed in 10 ml (Figs. 3B and 4B) or 200 μl (Fig. 3, D and G-J) of Buffer A (50 mm Tris-HCl, pH 8, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EGTA, 0.5 m sodium fluoride, phosphatase inhibitor mixture 2 (Sigma), Complete protease inhibitor mixture (Roche)), and cell extracts were cleared of membranous material by centrifugation at 10,000 × g for 15 min. Extracts were incubated with protein A-Sepharose 4B beads (Amersham Biosciences) for 1 h to reduce nonspecific binding of proteins to the beads in the subsequent purifications. Next, the pre-cleared extracts were incubated with 800 (Figs. 3B and 4B) or 80 μl (Fig. 3, D and G-J) of protein A-Sepharose beads and 10 (Figs. 3B and 4B) or 1 μl (Fig. 3, D and G-J) of the relevant antibody for 2 h. A rabbit anti-GFP antibody was used in the control purifications. Subsequently, beads were washed 3 times in Buffer A, boiled in sample buffer (Invitrogen), and resolved by SDS-PAGE on 8-16% gradient gels (Bio-Rad, Fig. 4B) or 4-12% NuPage BisTris gels (Invitrogen, Fig. 3, B, D, and G-J). Individual protein bands were visualized by Brilliant Blue G-colloidal concentrate (Sigma) staining, cut out, and identified by MALDI-TOF mass spectrometry at the Taplin Biological Mass Spectrometry Facility (Figs. 3B and 4B).FIGURE 4dMFAP1 forms a complex with several splicing factor homologues and is required for pre-mRNA processing. A, table summarizing proteins that were isolated in complex with endogenous dMFAP1. 1, when a protein does not have an obvious homologue in yeast, the fly and human IDs are indicated. B, to identify proteins that form a complex with dMFAP1, purifications were performed from S2 cells using anti-dMFAP1 or anti-GFP (control) antibodies. Eluates were resolved on a SDS-PAGE gel, and stained with Brilliant Blue G-colloidal concentrate. Visible bands (marked by asterisks) were excised and identified by MALDI-TOF mass spectrometry. The band corresponding to dMFAP1 is indicated by two asterisks. C, dMFAP1 is required for pre-mRNA processing. Total RNA was extracted from the S2 cells, and γ-tubulin mRNA (black) and pre-mRNA (gray) levels were measured after 1st strand cDNA synthesis by qPCR (see "Experimental Procedures"). mRNA and pre-mRNA levels were compared between the different samples by normalization to levels of the intronless his3 transcript.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GST pull-downs were performed using 1 μg of bacterially produced GST or GST-dMFAP1 protein with 50 ng of bacterially produced His-dPrp38 protein (Fig. 3E). His-dPrp38 pull-downs were performed using 500 ng of bacterial-produced His-dPrp38 or His-Peptide (control) and 800 ng of cleaved GST-dMFAP1 (Fig. 3F). Western Blotting—Proteins were resolved by SDS-PAGE using 4-12% gradient gels (Invitrogen) and transferred electrophoretically to polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were incubated for 1 h in Blocking Buffer (PBS (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.47 mm KH2PO4, pH 8), 5% milk), and incubated overnight at 4 °C in the same buffer containing primary antibodies at the following dilutions: anti-dPrp38_N, anti-dPrp38_C, anti-dMFAP1, 1:1000; anti-β-tubulin (Developmental Studies Hybridoma Bank), 1:2000; anti-GST (Cell Signaling Technology), 1:2000. Membranes were washed three times in PBS-T, blocked for 1 h, and probed with secondary antibodies diluted 1:5000 in Blocking Buffer for 1 h at room temperature. After three washes in PBS-T, chemiluminescence was observed using the ECL Plus Western blotting detection system (Amersham Biosciences). dsRNA—dsRNAs were synthesized with a Megascript T7 kit (Ambion). DNA templates for dsRNA synthesis were PCR amplified from fly genomic DNA or plasmids using primers that contained 5′T7 RNA polymerase-binding sites followed by sense or antisense sequences. The primers were designed using the E-RNAi at the DKFZ, Heidelberg (www.dkfz-heidelberg.de/signaling/ernai.html). For eGFP, the following primers were used: sense primer, GGTGGTGCCCATCCTGGT, antisense primer TCGCGCTTCTCGTTGGGG; for CG6049/cus2, sense primer, CTCCTCCTTTCTCTTGGCCT, antisense primer, CAAAACGGACGAAACTCCAT; for CG6905/cef1, sense primer, TCTCTAGCTCTCGCTTTCGG, antisense primer, AGCAGGTAGTCAAGCTGGGA; for CG8877/prp8, sense primer, TTGCTCCTTGGTCTGCTTTT, antisense primer, CATTCACACCTCTGTGTGGG; for CG32604/prp16, sense primer, TGTTCAGCAAGAACACCTGC, antisense primer, GCCGGAATCGATAACGTAGA; for CG6015/prp17, sense primer, CATTGATGTGGGCCTTCTCT, antisense primer, CACGCACCATCCCTAGTTTT; for CG6011/prp18, sense primer, GATGTCTCGCAGACTGTCCA, antisense primer, CTGAACGCCAAGAACACAGA; for CG5519/prp19, sense primer, AGCCAGATAGGTTCCGCTTT, antisense primer, ACAAACACTGGGCATTCTCC; for CG8241/prp22, sense primer, GCAGTCGGCTTTGTCTAAGG, antisense primer, ATGGTGTAGCCAACCTCCTG; for CG30342/dprp38, sense primer, AGCGTGTCTGCGACATTATACTGCCCC, antisense primer, AGCCTCGCGAGTCCCGTTCCC; and for CG1017/dmfap1, sense primer, AGGGAGCACAGGGAGCGATTCAGCGG, antisense primer, AGCATTCGCTTGAGTTCACGCAGCTTC. DNA Profiles—Cells were seeded in 35-mm wells at a density of 7 × 105 cells/ml in a total of 3 ml of complete medium/well and treated with dsRNA (20 μg/well) targeting genes encoding the indicated gene products for 3 days. Subsequently, cells were harvested, collected by centrifugation, washed two times in PBS, fixed in cold 70% ethanol, and stored at 4 °C. Subsequent steps were performed at room temperature. Fixed cells were washed twice in PBSA, treated with 50 μl of 100 μg/ml RNase A (Sigma) for 15 min and 250 μl of 40 μg/ml propidium iodide for a further 30 min, and then analyzed by flow cytometry. BrdUrd Pulse-Chase—Cells were seeded in 35-mm wells at a density of 7 × 105 cells/ml in a total of 3 ml of complete medium/well and treated with dsRNA (20 μg/well) targeting eGFP or dprp38 for 4 days. 15 μm BrdUrd (Sigma) was added to the medium for 15 min, then cells were washed three times with PBS, and BrdUrd-free medium was added. Cells were harvested at the indicated time points, collected by centrifugation, washed two times in PBS, fixed in cold 70% ethanol, and stored at 4 °C. Fixed cells were washed twice in PBS and once in PBS-BT (PBS + 0.1% bovine serum albumin + 0.2% Tween 20). 2 μl of monoclonal mouse anti-BrdUrd (BD Biosciences) were added directly to the cell pellets, incubated for 20 min in the dark, then cells were washed twice in PBS-BT and incubated in 50 μl of fluorescein isothiocyanate-conjugated rabbit antimouse F(ab′)2 fragments (DAKO) diluted 1:10 in PBS-BT for 20 min. Cells were washed twice in PBSA, treated with 50 μl of 100 μg/ml RNase A (Sigma) for 15 min and 250 μl of 40 μg/ml propidium iodide for a further 30 min and then analyzed by flow cytometry. Quantitative RT-PCRs—S2 were treated with dsRNA (20 μg/well) targeting genes encoding the indicated gene products for 3 days. Total RNA was isolated from the cells using the RNeasy kit (Qiagen) and treated with RQ1 DNase (Promega). Total RNA (1.5 μg) was used for first-strand cDNA synthesis with avian myeloblastosis virus reverse transcriptase and oligo-p(dT)15 primer (mRNA) or oligo-p(dN)6 (total RNA) (Roche). To measure pre-mRNA levels, quantitative PCRs (qPCRs) were performed on reverse-transcribed total RNA using one intron- and one exon-specific primer. To measure mRNA levels, qPCRs were carried out on reverse-transcribed total mRNA using exon-specific primers. For stg mRNA, sense primer, GCAGTTCTCCTTCTCAACGG, antisense primer, GGAGGAGCTGTCGTTCTACG; for γ-tubulin(23C) pre-mRNA, sense primer, GCGCCAAACCTACTATTAACTC, antisense primer, CTACATCACTGATCTCGTCCTG; for γ-tubulin(23C) mRNA, sense primer, GGCGGACGACGACCACTAC, antisense primer, GGATAGCGGTCCGCCAGGCGC; for eIF3-S10 pre-mRNA, sense primer, GCGGTGTCTGAAGAGAAAC, antisense primer, CCGCGGATTACATTTTCCAG; for eIF3-S10 mRNA, sense primer, GGCCCGCTATACGCAACGTC, antisense primer, CGGCCATTTTCAGGTAGCCG; for grt pre-mRNA, sense primer, GGAGGCATCAAGAATAACCG, antisense primer, GTTTCGATGCAAAAGGAGCTG; for grt mRNA, sense primer, GCAGCTGCGAGCACACTAATC, antisense primer, CTGGATAATTCTGGGAGGTGG; for hpo pre-mRNA, sense primer, GGAAAACGGAATGCAACAAC, antisense primer, CAATAACAAATGGCCAGCCCTTTC; for hpo mRNA, sense primer, CGGTGAATACCAACAGAGCTC, antisense primer, CGCCACGGCCATCTCCCGC; and for his3, sense primer, GTGAAGTAGTGAACGTGAAC, antisense primer, CCGCCGAGCTCTGGAATCGC. Real-time qPCR was performed with Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen). PCR was carried out in 96-well plates using the Chromo 4 Real-time qPCR Detection System (MJ Research). All reactions were performed in four replicates. The relative amount of specific mRNAs and pre-mRNAs under each condition was calculated after normalization to the intronless histone 3 (his3) transcript. dPrp38 is a nuclear protein required for developmental growth and proliferation. Budding yeast Prp38p belongs to a family of proteins defined by the presence of a conserved 180-amino acid Prp38 homology domain of unknown function (25Blanton S. Srinivasan A. Rymond B. Mol. Cell. Biol. 1992; 12: 3939-3947Crossref PubMed Scopus (68) Google Scholar). Homology searches of the Drosophila proteome revealed a protein encoded by the CG30342 gene, which contains a Prp38 homology domain at its N terminus (Fig. 1A). Overall, the Drosophila protein shares 75% identity with the human Prp38 protein (Prpf38a) and 22% identity with yeast Prp38p (17Chen Y.I. Maika S.D. Stevens S.W. J. Mol. Biol. 2006; 361: 412-419Crossref PubMed Scopus (7) Google Scholar) (supplemental Fig. S1). We will therefore refer to the CG30342-encoded protein as dPrp38. To characterize dPrp38 in vitro and in vivo, two antibodies were raised against the N and C termini of dPrp38. The antibodies were tested by Western blotting on extracts from cultured Drosophila S2 cells treated with dsRNAs targeting eGFP (control) or dprp38. In control-treated cells, both antibodies recognized a band of the expected size (35 kDa), which was greatly reduced upon RNAi treatment (Fig. 1B). As expected for a splicing factor, staining of cells in the salivary glands of a wild-type animal revealed that dPrp38 is localized exclusively in the nucleus (Fig. 1, C and D). To study dprp38 loss-of-function, we obtained a stock bearing a transposon (P-element) insertion in the open reading frame of dprp38 (dprp38G19491) (Fig. 1A). This insertion is predicted to give rise to a deletion of the last 65 amino acids of dPrp38, and a replacement of this region with two amino acids encoded by the P-element. Indeed, in Western blots of fly extracts from dprp38G19491 animals, we did not detect a product with our C-terminal antibody (Fig. 1E, middle panel), whereas the N-terminal antibody detected a product slightly above the wild-type band (Fig. 1E, top panel). The fact that the mutant band migrates higher than expected may be due to aberrant folding. Next, we used the FLP/FRT system (24Xu T. Rubin G.M. Development. 1993; 117: 1223-1237Crossref PubMed Google Scholar) to induce mitotic clones of dprp38G19491 mutant tissue in the eye-imaginal discs (the larval precursor of the adult eye) of heterozygous animals (Fig. 1, F and G). By inducing FLP/FRT-mediated recombination in early eye-imaginal discs, it is possible to generate homozygous mutant clones (no GFP), corresponding wild-type twin-spots (two copies of GFP), whereas heterozygous tissue has one copy of GFP (Fig. 1F). Gene dosage affects dPrp38 expression levels because the anti-dPrp38_C staining is brighter in homozygous wild-type than heterozygous tissue, whereas mutant tissue has little detectable staining (Fig. 1G). However, the dprp38G19491 insertion has no detectable effect on clone growth (compare the size of bright green and black areas in Fig
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