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C. Elegans GFP/RFP Alternative Splicing Screen

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C. Elegans GFP/RFP Alternative Splicing Screen ARTICLES Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation s d mechanisms in vivo o h t e 1,2 3,4 3,4 1,2 m Hidehito Kuroyanagi , Tetsuo Kobayashi , Shohei Mitani & Masatoshi Hagiwara e r u t a n / Alternative splicing of pre-mRNAs allows multicellular organisms native exons have been well characterized by analyzing model genes. m o c to create a huge diversity of proteomes from a finite number of Expression cloning strategies have permitted the global collection . e r genes. But extensive studies in vitro or in cultured cells have not of putative sequence elements that function in cultured cells, and u t a fully explained the regulation mechanisms of tissue-specific or bioinformatic analyses have identified putative cis elements within n . developmentally regulated alternative splicing in living exons and introns. Recently, however, conditional knockouts of w w organisms. Here we report a transgenic reporter system that trans-acting SR protein families revealed that alternative splicing of w / / : allows visualization of expression profiles of mutually exclusive only a few target genes are crucially dependent on a specific protein p t t exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A in cardiac muscles, even though many more genes expressed in this h and 5B showed tissue-specific profiles, and we isolated mutants 14 p tissue have typical cis elements . This indicates that we cannot u o defective in the tissue specificity. We identified alternative- precisely predict the alternative splicing patterns in each tissue r G splicing defective-1 (asd-1), encoding a new RNA-binding or cell without the assessment of the regulation mechanisms in g protein of the evolutionarily conserved Fox-1 family, as a living organisms. n i h regulator of the egl-15 reporter. Furthermore, an asd-1; fox-1 Some cell type–specific trans-acting alternative splicing factors s i l double mutant was defective in the expression of endogenous and their target genes have been identified in conventional genetic b u egl-15 (5A) and phenocopied egl-15 (5A) mutant. This studies on C. elegans and Drosophila melanogaster15,16, suggesting P e transgenic reporter system can be a powerful experimental feasibility of using the genetic approach for comprehensive studies r u t tool for the comprehensive study of expression profiles and on regulation of alternative splicing. As in vertebrates, most genes a 17 B N regulation mechanisms of alternative splicing in metazoans. in C. elegans have introns ,and 5% of protein-coding genes 6 0 have several different forms of their corresponding mature 0 18 2 The importance of the alternative splicing of pre-mRNAs on the mRNAs . Because of its advantage in morphological and genetic © structure and function of proteins, as well as on cellular processes, studies, we intended to generate a transgenic reporter system in has been discussed1–3. Recent global studies on cDNA sequences or C. elegans. Here we demonstrate that the reporter system allowed us microarray data have predicted that as many as two-thirds of to monitor the expression profiles of alternatively spliced exons at a human genes have multiple isoforms of mature mRNAs4,5,and single-cell resolution in vivo and can be used for identification of experiments with alternative-splicing microarrays revealed that cis-acting elements and trans-acting factors regulating the expres- many alternative splicing events are controlled in tissue- and cell sion of the reporter. We also demonstrate that regulation mechan- type– and/or developmental stage–dependent manners6,7.These isms identified using this reporter system can be applied to the findings indicate that unidentified ‘cellular codes’ underlie the endogenous gene in vivo, and that the regulation mechanisms of regulation of alternative splicing of many genes in living organ- tissue-specific alternative splicing recently characterized in verte- isms8–11. Experimental elucidation of the expression profiles and brates are also conserved in nematodes. regulation mechanisms of alternative splicing would lead to a better understanding of genome functions and cellular identities of RESULTS multicellular organisms. Generation of transgenic reporter worms Regulation mechanisms of alternative splicing have been experi- As a model gene, we used egl-15, which encodes the sole homolog mentally studied mostly in vitro and in cultured cells12,13.General of fibroblast growth factor receptor (FGFR) in C. elegans. cis-acting enhancer and silencer elements as well as trans-acting Its mutually exclusive exons 5A and 5B correspond to an insert factors involved in the regulation of both constitutive and alter- within the extracellular domain19. EGL-15(5B) and its ligand, 1School of Biomedical Science and 2Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 3Department of Physiology, Tokyo Women’s Medical University School of Medicine, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. 4CREST, JST, Hon-cho, Kawaguchi, Saitama 332-0012, Japan. Correspondence should be addressed to H.K. ([email protected]) or M.H. ([email protected]). RECEIVED 19 APRIL; ACCEPTED 24 AUGUST; PUBLISHED ONLINE 23 OCTOBER 2006; DOI:10.1038/NMETH944 NATURE METHODS | VOL.3 NO.11 | NOVEMBER 2006 | 909 ARTICLES Figure 1 | Alternative splicing reporter worms. (a) Construction of egl-15 a 200 bp egl-15 gene 3 4 5B 5A 6 reporter E5B-GFP–E5A-RFP (BGAR). Genomic fragment of egl-15 exon 4 through 5A was amplified, and cDNAs for GFP and RFP were inserted to Reporter cassette E4 GFP E5B E5A RFP construct the reporter cassette. The reporter cassette was introduced between Promoter unc-54 3' the promoter and 3¢ cassette by homologous recombination. Boxes indicate Expression vector BGAR E4 GFP E5B E5A RFP exons. (b) Schematic representation of mRNAs derived from the BGAR reporter in a. Constitution of mRNAs was analyzed by cloning and sequencing RT-PCR b mRNA E4/E5A E4 E5A-RFP products. Predicted ORFs are colored in green for E5B-GFP and in magenta E4/E5BE5A E4 E5B-GFP for E5A-RFP.*E5A,cryptic3¢-splice site within E5A. (c–f)Transgenicworms E4/E5B/ *E5A E4 E5B-GFP expressing BGAR reporter driven by unc-51 (c,d), myo-3 (e)anddpy-7 (f) promoters. Projection images of E5A-RFP, E5B-GFP, merged and DIC images of E5A-RFP E5B-GFP Merged DIC c the same fields are shown. (g) Schematic representation of BRAG mini-genes. (h–k) Merged views of confocal images of reporter worms expressing BRAG reporter under unc-51 (h,i), myo-3 (j)anddpy-7 (k) promoters. L2–L3 larvae s d (c,e,f,h,j,k). Lateral view of an adult vulva (d,i). Scale bars in c,e,f,h,j,k, o h 100 mm; in d,i,50mm. t unc-51p e m e d r u t 19 a unc-51p the specific expression of endogenous exon 5A in sex myoblasts , n / m e the vulval muscles, which derived from the sex myoblasts, exclu- o c . sively expressed E5A-RFP (Fig. 1d). When we interchanged the e r positions of cDNAs encoding GFP and RFP in the reporter mini- u t a gene (Fig. 1g), the expression patterns of GFP and RFP completely n . w myo-3p reversed (Fig. 1h,i), indicating that the tissue-dependent differen- w w tial expression of GFP and RFP was not due to the differential / / f : p stability of GFP- or RFP-encoding mRNAs or of these proteins in t t h different tissues. p Toanalyze expression profiles of the egl-15 reporter, we generated u o dpy-7p several transgenic worms with various tissue-specific promoters r G Promoter driving expression of the reporter mini-gene. The myo-3 promoter g E4 g BRAG RFP E5B E5A GFP n predominantly drove the expression of E5A-RFP in body-wall i E5A-GFP h muscles (Fig. 1e) and vulval muscles (data not shown). E5B-GFP s hiE5B-RFP j k i l b unc-51p was exclusively expressed in the hypodermis under the dpy-7 u P promoter (Fig. 1f). When we interchanged the positions of e r cDNAs encoding GFP and RFP (Fig. 1g), the expression patterns u unc-51p myo-3p dpy-7p t of GFP and RFP completely reversed (Fig. 1j,k). Further results a N summarized in Table 1 demonstrated that alternative splicing 6 0 of the egl-15 reporter was regulated in a tissue-dependent 0 2 LET-756(FGF), exert essential functions, whereas EGL-15(5A) manner in vivo. © is specifically expressed in sex myoblasts, and is required for the directed migration of the cells toward EGL-17(FGF)– Screening for mutants with altered expression profiles releasing cells19. To examine the applicability of our transgenic reporter system to To monitor the usage of mutually exclusive exons, we first study regulation mechanisms of alternative splicing in vivo,we introduced cDNAs for RFP and GFP downstream of exon 5A and screened for mutants defective in the tissue-specific expression of 5B, respectively, in separate mini-genes (Supplementary Note 1 the egl-15 reporter (Fig. 2). We mutagenized the transgenic worms online). When expressed under the unc-51 promoter, which drives that predominantly expressed E5A-RFP in body-wall muscles expression in a variety of tissues, the transgenic worms showed (Fig. 2c) and isolated worms expressing higher amounts of GFP differential expression patterns of RFP and GFP corresponding to than the parental strain using a fluorescence-assisted worm sorter alternative splicing patterns (see Supplementary Note 1 and (Fig. 2b). Of the B5 Â 104 genomes screened, we isolated 18 below). Next we constructed a unified reporter (Fig. 1), in which independent alleles with various expression profiles (Fig.
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