Article |
Correspondence to Shoichiro Ono: sono{at}emory.edu
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Abstract |
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Abbreviations used in this paper: ADF, actin depolymerizing factor; A/Q-rich, alanine- and glutamine-rich; EMSA, electophoretic mobility shift assay; RRM, RNA recognition motif.
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Introduction |
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Actin depolymerizing factor (ADF)/cofilin proteins enhance actin filament dynamics by severing filaments and accelerating monomer dissociation from the pointed ends of the filaments (Bamburg, 1999; Bamburg et al., 1999; Maciver and Hussey, 2002; Ono, 2003). Essential functions of ADF/cofilin in in vivo actin dynamics and cell viability have been demonstrated in several organisms (McKim et al., 1994; Gunsalus et al., 1995; Lappalainen and Drubin, 1997). In mammals, three ADF/cofilin isoforms are encoded by separate genes and expressed in different patterns of tissue distribution (Matsuzaki et al., 1988; Moriyama et al., 1990; Ono et al., 1994; Gillett et al., 1996; Thirion et al., 2001; Vartiainen et al., 2002). However, in the nematode Caenorhabditis elegans, the unc-60 gene undergoes alternative splicing and expresses two ADF/cofilin isoforms, UNC-60A and UNC-60B (McKim et al., 1994). Our previous studies have indicated that the two ADF/cofilin isoforms have different activities: UNC-60A strongly depolymerizes filaments, whereas UNC-60B binds to filaments with only weak depolymerizing activity (Ono and Benian, 1998; Ono, 1999; Mohri and Ono, 2003). More importantly, they are expressed in different tissues and required for specific actin-dependent processes: UNC-60A is expressed in nonmuscle cells and is required for embryonic cytokinesis (Ono et al., 2003), whereas UNC-60B is specifically expressed in the body wall muscle and regulates myofibril assembly (Ono et al., 1999). The unc-60 gene has nine exons and only the first exon is shared by unc-60A and unc-60B (McKim et al., 1994). Therefore, the tissue-specific expression of unc-60A or unc-60B is proposed to be determined by selection of the first splice acceptor site at the 5'-end of either exon 2A or 2B (McKim et al., 1994). However, the regulatory mechanism of tissue-specific splicing of the unc-60 pre-mRNA is unknown.
In this work, we report identification and characterization of a putative splicing factor that regulates muscle-specific splicing of the unc-60 pre-mRNA in C. elegans. We cloned SUP-12, a conserved RNA-binding protein, as a suppressor of unc-60B. sup-12 mutations strongly suppress the muscle defects of unc-60B mutants. This suppression is likely due to alteration in expression of the unc-60 splice variants in the muscle cells. SUP-12 localizes to the nuclei in body wall muscle and its RNA-binding domain directly binds to the unc-60 pre-mRNA in vitro. Our data support that SUP-12 is a novel member of tissue-specific regulators of alternative splicing.
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Results |
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Direct binding of SUP-12 to the unc-60 pre-mRNA
To determine whether the SUP-12 protein may be directly involved in pre-mRNA splicing of unc-60, we examined direct interaction between SUP-12 and unc-60 pre-mRNA in vitro by an electophoretic mobility shift assay (EMSA). Recombinant GST-tagged full-length SUP-12 protein or the COOH-terminal portion (residues 118248) of SUP-12 were poorly soluble (unpublished data) and therefore were not examined. However, the NH2-terminal portion (residues 1117) of SUP-12 containing the RRM domain was stable and soluble as a GST-fusion protein. When purified GST-SUP-12 (RRM) was incubated with various portions of in vitro transcribed unc-60 pre-mRNA (Fig. 6 A), it caused a band shift of only the 978-nt RNA fragment that encompasses the sequence of exon 1 to a portion of exon 5A (Fig. 6, A and B). GST alone did not cause a band shift of the RNAs (Fig. 6 B). These results indicate that the RRM domain of SUP-12 is sufficient for direct RNA-binding and so could confer specificity for the 5'-region of the unc-60 pre-mRNA.
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We then used shorter RNA fragments of 10152 bases in the pull-down assay with GST-SUP-12 (RRM) (Fig. 6, C and D) and characterized the interactions in a quantitative manner (Fig. 6, F and G). GST-SUP-12 (RRM) showed relatively strong binding with exon 1 (A1-1-1), the first intron (A1-1-2), and the second intron (A1-2-2) but did not significantly interact with exon 2A (A1-2-1; Fig. 6 F). Interestingly, an 18-nt truncation of A1-1-2 at the 3'-end (A1-1-2-UG; Fig. 6, C and D) weakened the interaction with GST-SUP-12 (RRM; Fig. 6, F and G). The truncated region contains repeats of UG that have been reported to bind to several RNA-binding proteins (Mittag, 1996; Takahashi et al., 2000; Buratti et al., 2004). The RNA oligonucleotide UG (5'-UGUGUGCCUG-3') strongly interacted with GST-SUP-12 (RRM; Fig. 6 E), whereas the oligonucleotide UC (5'-UCUCUCCCUC-3') showed nearly insignificant interaction (Fig. 6 E). Densitometric quantification of the results in Fig. 6 E indicates that only UG exhibited strong saturable binding with GST-SUP-12 (RRM; Fig. 6 F). Binding of GST-SUP-12 (RRM) to UG was saturated at a molar ratio of 1.1:1.0 with a dissociation constant of 0.31 µM, suggesting that they form a stoichiometric 1:1 complex with physiologically strong affinity. Removal of the UG repeats from A1-1-2 did not completely abolish the interaction of GST-SUP-12 (RRM) with A1-1-2-
UG (Fig. 6, F and G), suggesting that the UG repeats are sufficient but not necessary for this interaction. Binding of GST-SUP-12 (RRM) to A1-1-1, A1-1-2, or A1-2-2 did not reach saturation within the conditions used in this work (Fig. 6 G), so we were not able to determine stoichiometry and affinity. These results demonstrate that the RRM domain of SUP-12 directly interacts with the unc-60 pre-mRNA at multiple sites within exon 1 and the first and second introns. In particular, strong interaction of the SUP-12 RRM domain with the UG repeats near the 3'-splice site in the first intron supports that SUP-12 may function as a regulator of pre-mRNA splicing.
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Discussion |
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Our data indicate that, in muscle cells, SUP-12 normally inhibits production of the unc-60A mRNA, but it enhances expression of the unc-60B mRNA. Several possibilities for the mechanism by which SUP-12 regulates expression of unc-60A and unc-60B could be considered. The most probable model is that the general splicing machinery may preferentially induce the splicing between exons 1 and 2A to produce unc-60A, whereas SUP-12 likely acts as an inhibitor of this splicing event. This model is strongly supported by the presence of UG repeats near the 3'-end of the first intron, which strongly interacts with the RRM domain of SUP-12. In the human cystic fibrosis transmembrane conductance regulator pre-mRNA, the nuclear RRM protein TDP-43 binds to the UG repeats at the 3'-end of intron 8 and causes exon skipping (Niksic et al., 1999; Pagani et al., 2000; Buratti et al., 2004). Thus, SUP-12 and TDP-43 may negatively regulate splicing in a similar manner by binding to UG repeats and directly competing with the U2 auxiliary factor, which is an essential splicing factor that binds to 3'-splice sites (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999).
An alternative model is that SUP-12 may indirectly inhibit 3'-end processing of the unc-60A pre-mRNA. Because the SUP-12binding sites on the unc-60 pre-mRNA are not close to the polyadenylation site of unc-60A, SUP-12 may have to interact with 3'-end processing factors to influence this process. The last exon (5A) for unc-60A that contains the 3'-untranslated region resides upstream of exon 2B (Fig. 3 A). In nonmuscle cells, the mRNA 3'-end processing factors may cleave the pre-mRNA and polyadenylate after exon 5A but before exon 2B is transcribed. Therefore, in muscle cells, SUP-12 may inhibit 3'-end processing at exon 5A and promote elongation of the pre-mRNA. Indeed, in case of polycistronic genes in C. elegans, an interaction between a 3'-end processing factor and a factor for trans-splicing is reported (Evans et al., 2001). Also, it is possible that SUP-12 affects RNA stability or interacts with transcription factors and regulates transcription and pre-mRNA processing because splicing factors and transcription factors functionally interact and regulate pre-mRNA splicing in many instances (Bentley, 2002). In addition, we cannot exclude the possibility that SUP-12 may regulate relative stability of the two mRNAs.
The RRM domain of SUP-12 had activity to bind to the 5'-region of the unc-60 pre-mRNA but was unable to rescue the sup-12 mutant phenotype. This suggests that the COOH-terminal A/Q-rich sequence plays an important function. Although the function of the A/Q-rich sequence is unknown, it is intriguing that MEC-8, which regulates alternative splicing of unc-52 in the hypodermis in C. elegans (Lundquist et al., 1996; Spike et al., 2002), also contains an A/Q-rich region in addition to two RRM domains. The A/Q-rich sequence might have a regulatory function for a splicing factor or mediate interactions with other splicing factors, transcription factors, or 3'-end processing factors. We showed that the A/Q-rich region of SUP-12 is necessary and sufficient for speckled localization in the nuclei, suggesting that this region is important for SUP-12 to localize to speckles. Also, we noted that the bacterially expressed full-length SUP-12 protein was not only insoluble but also very susceptible for proteolysis (unpublished data), suggesting that the A/Q-rich sequence may regulate protein stability.
Furthermore, this work suggests that functional redundancy of the two ADF/cofilin isoforms in muscle is normally masked by tight regulation of tissue-specific splicing by sup-12. Thus, sup-12 mutations unmask the redundancy and allow UNC-60A to compensate for mutated UNC-60B in unc-60B mutants. From our previous work, it seemed logical to hypothesize that UNC-60B with weaker depolymerizing activity might be more suitable in muscle cells than UNC-60A, where less dynamic actin reorganization is needed than nonmuscle cells. Therefore, it is somewhat surprising that UNC-60A can substitute for UNC-60B in muscle. However, it is possible that, although the sup-12 mutants have apparently normal myofibrils, their muscle may exhibit different physiological properties from that of wild type under specific conditions. A number of human diseases are caused by alterations in the pre-mRNA splicing (Nissim-Rafinia and Kerem, 2002; Stoilov et al., 2002; Faustino and Cooper, 2003). However, our results suggest that, the splicing machinery is a potential therapeutic target for certain genetic diseases in which manipulation of tissue-specific splicing machinery may reveal hidden functional redundancy among splice variants to compensate for a disease gene. We propose that SUP-12 and SEB-4related proteins are a new family of tissue-specific splicing factors in multicellular organisms.
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Materials and methods |
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Cloning of sup-12
sup-12 was mapped to the left arm of the X chromosome that was included in the duplication mnDp33 (G.R. Francis and R.H. Waterston, personal communication). We further narrowed down sup-12 by the snip-SNP mapping with polymorphisms in CB4856 (Wicks et al., 2001) to an interval between cosmid clones ZC64 and T06F4 that contained 20 genes. We performed feeding RNA interference of 10 genes in wild type and unc-60B(su158) and examined for a suppressor phenotype for unc-60B(su158). We found that T22B2.4(RNAi) suppressed the motility defect of unc-60B(su158) but did not affect motility of wild type. To confirm that T22B2.4 is sup-12, we sequenced the T22B2.4 gene in the sup-12 mutants and identified mutations in multiple sup-12 alleles (Results and Fig. 2 A).
Fluorescence microscopy
Actin filaments were visualized by staining adult worms with tetramethylrhodamine-phalloidin (Sigma-Aldrich) as described previously (Ono, 2001). Immunofluorescent staining was performed on adult worms that were permeabilized with a freeze-crack method (Epstein et al., 1993) and fixed with methanol for 5 min at 20°C. Primary antibodies used were antiUNC-60A (Ono et al., 1999) and anti-myoA (mAb 5.6, obtained from H.F. Epstein; Miller et al., 1983). Secondary antibodies were Alexa488-labeled goat antirabbit IgG and Alexa647-labeled goat antimouse IgG (Molecular Probes). To visualize nuclei, worms were fixed with 4% formaldehyde in PBS for 30 min at RT, permeabilized with acetone at 20°C for 5 min, and stained with DAPI (Sigma-Aldrich) at 1 µg/ml in PBS containing 0.5% Triton X-100, 1 mM EDTA, and 0.05% sodium azide for 15 min.
Fluorescent samples were mounted with the ProLong antifading reagent (Molecular Probes) and viewed by epifluorescence using an inverted microscope (model Eclipse TE2000; Nikon) with a 40x CFI Plan Fluor objective (dry; NA 1.4). Images were captured by a SPOT RT Monochrome CCD camera (Diagnostic Instruments) and processed by the IPLab imaging software (Scanalytics, Inc.) and Adobe Photoshop 6.0.
Northern and Western blots
Total nematode RNA was isolated using a TRI reagent (Sigma-Aldrich). RNA samples (10 µg) were subjected to formaldehyde-agarose gel electrophoresis, transferred to positively charged nylon membranes (Millipore), and fixed by ultraviolet irradiation. cDNAs for unc-60A (505 bp), unc-60B (460 bp), and actin (act-1) (1.1 kb) were amplified by PCR, labeled with digoxigenin with random priming, and used as probes for Northern blotting using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science). Western blot was performed as described previously (Ono and Ono, 2002) using the following primary antibodies: antiUNC-60A (Ono et al., 1999), antiUNC-60B (Ono et al., 1999), anti-actin monoclonal C4 (ICN Biomedicals), and anti-tubulin (Amersham Biosciences).
Transgenic expression of GFP and GFP-SUP-12
To determine the promoter activity of sup-12, a 3,418-bp genomic fragment containing the 3,087-bp upstream region, exon 1, intron 1, and 58-bp of exon 2, was amplified by PCR using ExTaq DNA polymerase (Takara) and cloned into the gfp expression vector pPD95.67 (obtained from A. Fire, Stanford University, Stanford, CA) at the 5'-end of the gfp coding region. For expression of GFP-SUP-12 in body wall muscle, the sup-12 cDNA (yk1125e08, obtained from Y. Kohara, National Institute of Genetics, Mishima, Japan) was ligated in-frame with the 3'-end of gfp in pPD118.20 (obtained from A. Fire) that has the myo-3 promoter. For expression of fragments of SUP-12 as GFP-fusion proteins, fragments of the sup-12 cDNA encoding residues 1117 or 118248 were amplified by PCR and ligated in-frame with the 3'-end of gfp in pPD118.20. A synthetic stop codon was added for expression of residues 1117. The plasmids were injected into hermaphroditic gonads of wild type or unc-60B(su158);sup-12(st89) at 10 µg/ml together with a dominant marker pRF4(rol-6(su1006)) at 90 µg/ml, and transgenic worms were selected by their roller phenotype or expression of GFP.
Electrophoretic mobility shift assay
A cDNA fragment encoding residues 1117 of SUP-12 and a synthetic stop codon was amplified by PCR and cloned into the SmaIEcoRI cloning site of a GST expression vector pGEX-2T (Amersham Biosciences). The insert was sequenced to confirm that no mutations were introduced by PCR. GST alone or GST-SUP-12 (RRM) was expressed in Escherichia coli BL21 (DE3) by induction with 0.1 mM IPTG for 3 h and purified with Glutathione-Uniflow (BD Biosciences Clontech) following manufacturer's instruction.
Template DNA fragments used in the in vitro transcription reactions were amplified from C. elegans genomic DNA using the primers listed in Tables S1 and S2 (available at http://www.jcb.org/cgi/content/full/jcb.200407085/DC1). The sense primers contain the SP6 promoter sequence. Each RNA was in vitro transcribed using the MAXIscript kit (Ambion) in the presence of 50 µCi of -[32P]CTP (Amersham Biosciences) and purified with a Sephadex G-25 spin column (Amersham Biosciences). RNAs (5 x 105 cpm) were denatured in water at 95°C for 2 min, chilled on ice for 2 min, and incubated with either buffer, or buffer and 1 µM GST or buffer and 1 µM GST-SUP-12 (RRM) for 30 min at RT. The buffer contained 50 mM KCl, 5 mM MgCl2, 10% glycerol, 40 mM DTT, 0.1 mg/ml yeast tRNA, and 10 mM Hepes, pH 7.4). The samples were resolved on a 1% agarose gel in 1 x TAE (40 mM Tris-acetate, 2 mM EDTA, pH 8.3). The gels were dried and analyzed by the PhosphorImager (Molecular Dynamics).
Biotin-RNA pull-down assay
Biotin-RNA pull-down assay (Lee and Schedl, 2001) was performed with the following modifications. Template DNAs for in vitro transcription of RNAs (95284 bases) were amplified from C. elegans genomic DNA using the primers listed in Tables S1 and S2. The sense primers contain the T7 promoter sequence. Biotin-labeled RNAs were in vitro transcribed with T7 RNA polymerase (Invitrogen) in the presence of Biotin RNA Labeling Mix (Roche Applied Science) at 37°C for 2 h. The template DNAs were digested by RNase-free DNase I (Roche Applied Science) and the labeled RNAs purified with SigmaSpin Post-Reaction Clean-Up Columns (Sigma-Aldrich). RNA oligonucleotides UG (5'-UGUGUGCCUG-3') and UC (5'-UCUCUCCCUC-3') were synthesized and chemically labeled by biotin at the 5'-ends by Integrated DNA Technologies.
Biotin-labeled RNA at 0.1 µM was incubated with GST or GST-SUP-12 (RRM) in RP buffer (50 mM KCl, 1 mM MgCl2, 10 mM Hepes-NaOH, pH 7.5) containing 10 mM DTT and 100 µg/ml yeast tRNA (Ambion) in a final volume of 200 µl at RT for 30 min. The mixtures were incubated with 0.1 mg of streptavidin MagneSphere Paramagnetic Particle (Promega) at RT for 20 min. The magnetic particles were isolated with a magnetic separation stand, washed three times with RP buffer, suspended in 20 µl of SDS sample buffer (2% SDS, 80 mM Tris-HCl, 5% ß-mercaptoethanol, 15% glycerol, 0.05% bromophenol blue, pH 6.8), and incubated at 97°C for 2 min. Bound proteins were analyzed by SDS-PAGE (12% acrylamide gel) and staining with Coomassie Brilliant blue R-250 (National Diagnostics). The Coomassie-stained gels were scanned by a UMAX PowerLook III scanner at 300 dots per inch and the band intensity was quantified with Scion Image Beta 4.02 (Scion Corporation) by comparing with the intensities of known amounts of GST-SUP-12 (RRM).
Online supplemental material
Table S1 contains a list of PCR primers for amplifying template DNAs for in vitro transcription. Table S2 contains a list of primer combinations for PCR amplification of the template DNAs. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200407085/DC1.
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Acknowledgments |
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Some preliminary data were obtained by S. Ono while he was a postdoctoral fellow in the laboratory of Guy Benian, which was supported by a grant from the National Science Foundation (MCB-9728762). Some worm strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources. This work was supported by grants from the NSERC Canada to R.C. Johnsen and D.L. Baillie, the American Heart Association Southeast Affiliate, and the National Institutes of Health (R01 AR048615) to S. Ono.
Submitted: 13 July 2004
Accepted: 12 October 2004
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