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RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3′ UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that breakdown and dedifferentiate back to single, mitotically active cells. Together these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox related diseases.
RNA binding proteins play an integral role in mRNA metabolism, splicing, transport and translation. An increasing number of studies link mutations in genes encoding RNA binding proteins with a variety of diseases, highlighting the importance of these proteins in regards to human health (Lukong et al., 2008; Ramaswami et al., 2013). Rbfox proteins represent one such family and contain a highly conserved, centrally located RNA-recognition motif (RRM) flanked by intrinsically disordered regions (IDRs) (Auweter et al., 2006; Jin et al., 2003; Ponthier et al., 2006). Mammals have three Rbfox paralogs: RBFOX1 (A2BP1), RBFOX2 (RBM9) and RBFOX3 (NeuN) (Kim et al., 2009; Kuroyanagi, 2009). Nuclear isoforms of these genes regulate alternative splicing by directly binding to intronic (U)GCAUG elements, resulting in the exclusion or inclusion of downstream or upstream exons respectively. In mice, disruption of Rbfox1 in neurons leads to neuronal hyperactivity while loss of Rbfox2 results in cerebellum development defects (Gehman et al., 2012; Gehman et al., 2011). Rbfox1 and Rbfox2 has been implicated in a number of diseases including cancer, diabetes and neurological disorders such as autism, mental retardation and epilepsy (Barnby et al., 2005; Bhalla et al., 2004; Davis et al., 2012; Mikhail et al., 2011; Sebat et al., 2007). In all these examples, the observed phenotypes have been attributed to perturbations in normal mRNA splicing patterns.
Specific isoforms of Rbfox genes localize to the cytoplasm of cells in a variety of tissues across species (Dredge and Jensen, 2011; Gehman et al., 2012; Hamada et al., 2013; Kiehl et al., 2001; Lee et al., 2009; Shibata et al., 2000). While the molecular functions of these isoforms remain poorly understood, both nuclear and cytoplasmic isoforms appear to act as tumor suppressors in the context of glioblastomas (Hu et al., 2013). Loss of cytoplasmic Rbfox1 has also been associated with colorectal cancer (Sengupta et al., 2013) and abnormal cytoplasmic inclusions of Rbfox1 are often observed in SCAII patients (Shibata et al., 2000). Recent studies have also showed that Rbfox proteins bind to many different 3′UTRs in the mammalian brain (Lee et al., 2016; Weyn-Vanhentenryck et al., 2014). These observations suggest that Rbfox proteins carry out additional functions beyond their established roles in splicing.
The Drosophila genome contains a single Rbfox homolog called A2bp1, hereafter referred to as Rbfox1 to remain consistent with nomenclature across species. Mutations in the Drosophila Rbfox1 result in germline tumor formation (Tastan et al., 2010). Here, we show that cytoplasmic Rbfox1 is necessary for Drosophila germline development (Fig. 1A) and regulates the stability and translation of specific mRNAs by binding to (U)GCAUG elements contained within their 3′UTR sequences. We further show that within the germline Rbfox1 targets pumilio to promote differentiation. Thus, our study reveals a splicing-independent function of Rbfox proteins, the disruption of which may contribute to RBFOX-linked diseases.
Current annotations indicate that the Drosophila Rbfox1 gene encodes at least eight different isoforms. All of the corresponding proteins contain a nuclear localization signal (NLS) in their C-termini, except for the Rbfox1-PF isoform (Fig. 1B). We cloned a cDNA corresponding to an additional transcript (Rbfox1-RN) that also lacks an NLS from ovarian RNA. Antibodies directed against sequences shared by all nine Rbfox1 isoforms showed that while many tissues express nuclear Rbfox1, early developing germline cysts display a burst of cytoplasmic Rbfox1 protein expression (Tastan et al., 2010) (Fig. 1C). We anticipated that this staining reflects the expression of Rbfox1-PF and Rbfox1-PN. To begin to test this, we generated transgenic lines carrying HA tagged Rbfox1-RF or Rbfox1-RN cDNA expression constructs and found that the corresponding proteins localized to the cytoplasm in both germ cells and somatic cells, as expected (Fig. 1D; Fig. S1). Null Rbfox1 mutations result in lethality, whereas hypomorphic mutations result in female sterility and a germ cell tumor phenotype, marked by the accumulation of germline cysts that fail to differentiate beyond the early stages of their development (Tastan et al., 2010) (Fig. 1E). Expression of transgenes for either cytoplasmic isoform rescued the tumorous phenotype associated with the Rbfox1e03440 allele (Fig. 1F; Fig. S1E). We then generated two inducible shRNA lines targeting different regions of the exon sequence unique to the Rbfox1-RF and Rbfox1-RN transcripts. When expressed in the germline, these shRNA constructs caused a tumorous phenotype, mimicking the defects observed in Rbfox1 mutants (Fig. 1G; Fig. S1F,G). A CRISPR/Cas9 strategy was also used to delete the Rbfox1-RF and Rbfox1-RN specific exon (Fig. S1J). This mutant, Rbfox1dsRed.1, exhibited loss of cytoplasmic Rbfox1 expression during early germline cyst development, whereas nuclear Rbfox1 isoforms appeared largely unaffected (Fig. 1H; Fig. S1). This allele resulted in female sterility marked by the formation of tumors comprised of undifferentiated germ cells (Fig. 1H; Fig. S1K,L). Somatic cells of Rbfox1dsRed.1 mutant ovaries did not display any obvious phenotype. These experiments indicate that cytoplasmic Rbfox1 isoforms promote early germline cyst differentiation.
Drosophila Rbfox1 contains a highly conserved RNA Recognition Motif (RRM), which shares extensive amino acid identity with its human homolog. Rbfox1 function during early germline development depended on this RRM domain (Fig. S2A–C). Given the shared sequence homology, we hypothesized that Drosophila Rbfox1 binds to the same (U)GCAUG element, in which the (U) residue can vary, as its mammalian homologs. To test this, we performed RNA-EMSA experiments and found that recombinant Drosophila Rbfox1 RRM associates with in vitro transcribed RNA that contains (U)GCAUG elements. By contrast, the Drosophila Rbfox1 RRM domain did not bind to RNA containing (U)GCAUA elements (Fig. 2A).
With rare exceptions, most splicing takes place within the nucleus (Braunschweig et al., 2013). Therefore, we hypothesized that cytoplasmic Rbfox1 associates with (U)GCAUG elements to carry out splicing independent functions within the Drosophila germline. We considered the possibility that cytoplasmic Rbfox1 regulates the stability or translation of specific mRNAs through a 3′UTR dependent mechanism. We engineered a series of different sensors that contain either one, two or three (U)GCAUG elements (1X Rbfox1 sensor, 2X Rbfox1 sensor or 3X Rbfox1 sensor respectively) or 3X (U)GCAUA elements (mutant sensor) embedded in α-Tubulin (α-Tub) 3′UTR (Fig. 2B). These reporters lacked introns, avoiding any complication that might result from the regulation of splicing. We then performed RNA-immunoprecipitation (RNA-IP) from cross-linked tissue extracts to verify that cytoplasmic Rbfox proteins associated with the 3X Rbfox1 sensor mRNA in vivo (Fig. 2C). We next determined if Rbfox1 regulates the expression of these reporters in vivo. The 1X Rbfox1 sensor was expressed throughout the germarium (Fig. 2D,D′), while the 2X Rbfox1 and 3X Rbfox1 sensors exhibited decreased expression in Rbfox1 expressing germ cells (Fig. 2E–F′). By contrast, the mutant sensor with three (U)GCAUA sites did not display reduced expression in Rbfox1 positive cells (Fig. 2G,G′). Examination of the 3X Rbfox1 sensor in an Rbfox1 mutant background confirmed that the presence of Rbfox1 was responsible for the observed decrease in reporter expression (Fig. 2H–H′). We constructed similar 3′UTR reporters using the gal4-UAS system and found that cytoplasmic Rbfox1 also repressed expression of mRNAs that contain (U)GCAUG elements within their 3′UTR in the nervous system (Fig. S2D–K). A combination of quantitative RT-PCR (qRT-PCR) and Poly(A) Tail-Length (PAT) assays using the 3X Rbfox1 and mutant sensors as read-outs showed the presence of (U)GCAUG sequences does not induce obvious changes in reporter transcript stability or poly-A tail length (Fig. S2L–P), suggesting that cytoplasmic Rbfox1 can block gene expression at the level of translation.
In order to identify endogenous cytoplasmic Rbfox1 target genes within the germline, we compared the conservation of core GCAUG sites, with or without the variable 5′ (U) residue, within mRNA 3′UTRs across different Drosophila and insect species. This analysis revealed that the short 1.2 kb pumilio 3′UTR, which exhibits enriched ovarian expression (flybase.org), contains four GCAUG sites. Two of these are absolutely conserved across 12 and 13 species respectively (Fig. 3A). pumilio stood out as a potentially significant Rbfox1 target mRNA: Pumilio regulates germline stem cell (GSC) maintenance by repressing the translation of specific mRNAs, and loss of pumilio results in precocious germ cell differentiation (Forbes and Lehmann, 1998; Slaidina and Lehmann, 2014). RNA-IP experiments reveal that Rbfox1 associates with pumilio mRNA in vivo, albeit at low levels (Fig. S3F).
We found Pumilio expression decreases in the presence of cytoplasmic Rbfox1 in wild-type germ cells and loss of cytoplasmic Rbfox1 results in increased Pumilio protein expression within the germline (Fig. 3B,C; Fig. S3). Next we compared the expression levels of pumilio mRNA and protein in ovaries carrying a synchronized population of germ cells in the presence or absence of Rbfox1 (Fig. 2L–M). To perform this experiment we crossed Rbfox1 mutations into a hs-bam; bamΔ86 homozygous mutant background. Loss of bam prevents germ cell differentiation, leading to the formation of large tumors that contain germ cells arrested in a pre-cystoblast like state. Heat-shock induction of the bam rescuing transgene causes all the germ cells within these tumors to undergo synchronous differentiation. By waiting a set period of time, we can isolate ovaries highly enriched for germ cells at a specific stage of development. Using this genetic background allows us to avoid complications associated with assaying a mixed population of germ cells at different stages of differentiation. qRT-PCR analysis of samples derived using this system revealed that pumilio mRNA levels increased in the absence of Rbfox1 (Fig. 3D). However, the levels of Pumilio protein increased to a much greater degree in the absence of Rbfox1 (Fig. 3E), suggesting that while Rbfox1 may destabilize pumilio mRNA, it also represses pumilio mRNA translation.
To test the functional significance of increased Pumilio expression in Rbfox1 mutant ovaries, we examined whether RNAi knockdown of pumilio modified the phenotype caused by RNAi knockdown of cytoplasmic Rbfox1. These experiments revealed that loss of pumilio strongly suppressed the germ cell tumor phenotype of Rbfox1-RFRNAi ovaries, resulting in the formation of egg chambers containing nurse cells with polyploid nuclei (Fig. 3F–H). Control experiments showed that this suppression was not due to changes in Rbfox1 expression levels (Fig. S3B–D). These data suggest that pumilio represents a functionally significant in vivo target of cytoplasmic Rbfox1-dependent gene regulation.
To determine whether the decrease in Pumilio expression in Rbfox1 expressing cells depends on the GCAUG elements within the pumilio 3′UTR, we constructed two reporter constructs: a wild-type pumilio 3′UTR reporter and a mutant pumilio 3′UTR reporter in which all four of the GCAUG elements were changed to ACAUA (Fig 3I). The wild-type pumilio 3′UTR reporter exhibited the same decreased expression in Rbfox1 expressing cells as Pumilio protein, suggesting that Pumilio expression is controlled, at least in part, in a 3′UTR-dependent manner (Fig 3J–J′; Fig S3E–E″). By contrast, the mutant reporter was expressed throughout the early germline, and actually displayed a marked increase in many Rbfox1 expressing cysts (Fig. 3K–K′). Both the wild-type and mutant pumilio 3′UTR reporters exhibited continuous expression in Rbfox1 mutant germaria (Fig. S3G–H).
We next tested whether low or high levels of ectopic pumilio expression disrupted normal germ cell differentiation using two transgenes containing full-length pumilio coding sequence and either endogenous pumilio 3′UTR (pum-pum) or α-Tubulin 3′UTR (pum-Tub) (Menon et al., 2004) (Fig. 4A; Fig. S4). Germline expression of the pum-pum transgene resulted in a mild phenotype, whereas expression of the pum-Tub transgene completely blocked germline cyst differentiation, resulting in a tumorous phenotype that strongly resembled the Rbfox1 mutant phenotype (Fig. 4B–E). Interestingly, many of the posteriorly positioned germ cells exhibited signs of dedifferentiation, a process previously described in both the Drosophila ovary and testis (Brawley and Matunis, 2004; Kai and Spradling, 2004; Sheng et al., 2009) (Fig 4F–H; Fig. 5). For example, germ cells expressing the pum-Tub 3′UTR transgene re-acquired high levels of cytoplasmic Sxl, which typically marks GSCs, cystoblasts and 2-cell cysts (Fig. 4F–H). These cysts also appeared to break down into single cells, as marked by changes in fusome and ring canal morphology (Fig. 5; Fig. S5). These single dedifferentiated germ cells remained mitotically active, as reflected by phospho-histone H3 staining, and the incidence of cell death appeared similar in control germaria and those over-expressing pumilio (Fig. S5). Re-examination of Rbfox1dsRed.1 mutant ovaries revealed that loss of cytoplasmic Rbfox1 also resulted in breakdown and dedifferentiation of multicellular cysts (Fig. 5). These results suggest a model whereby Rbfox1 promotes differentiation, in part, by repressing Pumilio expression. Cytoplasmic Rbfox1 activity provides a genetic barrier that prevents the inappropriate reversion of germline cysts back to an earlier development state (Fig. 5F).
Rbfox family members from different species localize to either the nucleus or the cytoplasm. While isoforms that localize to the nucleus play a clear role in regulating alternative splicing (Gehman et al., 2011; Hamada et al., 2013; Lee et al., 2009), the function of cytoplasmic isoforms has remained less clear. Previous results showed that loss of Rbfox1 in Drosophila resulted in a block of germ cell differentiation (Tastan et al., 2010). Here we sought to determine the extent to which disruption of either nuclear or cytoplasmic Rbfox1 isoforms contributed to this phenotype. Transgenic rescue, isoform specific RNAi knockdown and isoform specific knockout experiments provide strong evidence that two cytoplasmic Rbfox1 isoforms specifically promote germ cell differentiation during the early stages of germline cyst development. The Drosophila genome does not encode for another redundant Rbfox family member. Thus the Drosophila ovary represents a unique platform on which to explore the function of cytoplasmic Rbfox family members in an in vivo setting.
Further experiments showed that cytoplasmic Rbfox1 regulates gene expression through a 3′UTR-dependent mechanism. The defining RRM domain of the Rbfox protein family is highly conserved across species. In vitro and in vivo experiments presented here indicate that Drosophila Rbfox1 physically associates with RNAs that contain GCAUG elements, similar to mammalian Rbfox proteins. Recent studies using RNA-CLIP approaches have shown that mammalian Rbfox1, Rbfox2 and Rbfox3 all physically interact with 3′UTR sequences that contain GCAUG sites or other similar elements (Weyn-Vanhentenryck et al., 2014). Our experiments show that the presence of GCAUG sites within mRNA 3′UTRs results in modest decreases in mRNA stability, and more much dramatic decreases in protein expression. These observations suggest that Drosophila Rbfox1 acts to repress the translation of specific target mRNAs.
We observed that an increasing number of GCAUG sites within 3′UTRs appeared to have an additive effect on target gene expression in the context of germ cells. The presence of one site had little or no effect, at least in the context of our reporters, while the presence of two or three sites resulted in a clear repression of protein expression in Rbfox1 expressing cells. While the repression of 3′UTR GCAUG reporters occurred in both the germline and within specific neurons, it remains possible that cytoplasmic Rbfox family members may regulate gene expression in a different manner in different contexts. For example, a newly published study shows that mammalian Rbfox proteins can promote the stability and translation of a target gene in cell culture (Lee et al., 2016). The functional significance of this regulation remains to be tested in vivo. Regardless, these findings, together with results presented here, indicate that the ability of cytoplasmic Rbfox family members to regulate protein expression has been conserved across species. The direction and degree of cytoplasmic Rbfox-dependent gene regulation may depend on different cell specific proteins or on the presence of other 3′UTR regulatory elements within a given target transcript. The discovery of this function has significant implications in our understanding of how Rbfox family members regulate normal development, as well as the disorders linked with disruption of Rbfox genes such as epilepsy, autism and cancer.
Our search for functionally relevant endogenous mRNA targets of Drosophila Rbfox1, led to the finding that Rbfox1 represses Pumilio protein expression during early germline cyst differentiation. Previous studies noted the presence of Pumilio protein in germline stem cells, cystoblasts and 2-cell cysts (Forbes and Lehmann, 1998), but the mechanisms responsible for the stage specific decrease of Pumilio expression in 4-, 8- and 16-cell cysts, and the functional significance of this expression pattern, has remained unknown. Here we show that Pumilio expression decreases as Rbfox1 expression increases. Examining the 3′UTR sequence of pumilio revealed the presence of four GCAUG sites, two of which showed extensive sequence conservation across many Drosophila species. Strikingly, Rbfox1, Rbfox2 and Rbfox3 also physically associate with Pumilio1 and Pumilio2 mRNA in the mouse nervous system (Weyn-Vanhentenryck et al., 2014).
Further analysis showed that Pumilio expression in the germline is regulated through a 3′UTR dependent mechanism. A wild-type pumilio 3′UTR reporter exhibited an expression pattern similar to the endogenous protein, displaying decreased expression in the presence of Rbfox1. Mutating each of the four GCAUG elements within the pumilio 3′UTR sequence resulted in a striking expansion of reporter expression into the 4-, 8- and 16-cell cyst stages, suggesting that Rbfox1 negatively regulates Pumilio expression. Quantitative RT-PCR analysis of synchronously differentiating germ cells showed that endogenous pumilio mRNA levels increased in the absence of Rbfox1. These findings are in contrast to data obtained comparing the 3X Rbfox1 sensor to the mutant reporter, and suggest that Rbfox1 may influence the stability of specific target mRNAs in different contexts. Nonetheless, the degree to which Pumilio protein expression increases in the absence of Rbfox1 in these experiments is consistent with the model that Rbfox1 also regulates the expression of Pumilio, at least in part, at the level of translation. Other translational regulators, such as Bruno, also appear to influence mRNA stability (Kim et al., 2015).
The repression of Pumilio expression by Rbfox1 helps to promote germ cell differentiation. Loss of Rbfox1 results in germline tumor formation and an expansion of Pumilio expression. Strikingly, knockdown of pumilio strongly suppresses the Rbfox1 tumorous phenotype, leading to the formation of egg chambers with polyploid nuclei. While cytoplasmic Rbfox1 likely regulates the expression of other genes, the strength of this genetic interaction indicates that pumilio represents a major functional target of Rbfox1 in regards to germ cell differentiation. Mis-expression of a pumilio transgene, lacking the endogenous 3′UTR, in an otherwise wild-type background, phenocopies Rbfox1 mutants. These data indicate that germ cells must repress Pumilio expression before they can proceed into the next stage of development. Pumilio homologs are essential genes for germ cell maintenance across species. Given the conservation of Rbfox1 binding sites within the 3′UTR of pumilio mammalian homologs, repression of pumilio by Rbfox may represent a conserved mechanism that promotes germline differentiation.
Strikingly, morphological and molecular markers suggest that over-expression of Pumilio results in the dedifferentiation of germ cells. Rbfox1 mutants exhibit a similar phenotype. Pumilio over-expressing ovaries contain large tumors with multicellular cysts throughout their germaria. As these cysts continue to age and move towards the posterior of germaria, they begin to breakdown, as marked the fragmentation of fusomes and ring canals. Similar observations have been made in both the Drosophila ovary and testis when germline cysts are experimentally prompted to undergo dedifferentiation (Brawley and Matunis, 2004; Kai and Spradling, 2004). Furthermore, these germ cells reacquire the expression of cytoplasmic Sxl, which typically marks germline stem cells, cystoblasts and 2 cell cysts (Chau et al., 2009, 2012). Single cells derived from cyst breakdown remain mitotically active. These data indicate that germ cells must actively shut down gene expression programs that foster self-renewal and early differentiation before they can advance to the next stage of development. Failure to do so results in the reversion of the cells back to an earlier developmental state. We anticipate that loss of Rbfox1, and the corresponding mis-expression of Rbfox1 target genes, may have similar effects in different tissues and in different species.
Fly stocks were maintained at 20–25°C on standard c ornmeal–agar–yeast food. Vasa-gal4 was a gift from Y. Yamashita. Df(3L)ED4457, GH146-gal4 and LN1-gal4 and TRiP.HMS01564 (pumRNAi) were obtained from Bloomington Stock Center. Rbfox1e03440 and Rbfox1f02600 were obtained from Harvard Stock Center. UAS-pum-pum3′UTR and UAS-pum-Tub3′UTR were a gift from Elizabeth Gavis and originally described in Menon et al (2004). UAS-Rbfox1-RF was inserted into ZH-51D (BL#24483), UAS-Rbfox1-RN into ZH-22A (BL#24481), recombineered Rbfox1[FF>AA] and Rbfox1[WT] genomic constructs into VK00037 (BL#24872), all Rbfox, control sensors and pum 3′UTR reporters into ZH-51D (BL#24483) and shRNA constructs into attP6 (BL#34768) using phiC31 integrase (Rainbow Transgenics Inc.).
Adult ovaries were stained according to (Tastan et al., 2010). Drosophila adult brains were stained according to (Das et al., 2011). The following primary antibodies were used: guinea pig anti-Rbfox1 (1:5000) (Tastan et al., 2010), rat anti-Pum (1:1000) (Joly et al., 2013), mouse anti-Hts (1B1) (1:20), rat anti-VASA (1:20), rabbit anti-GFP (1:1000) (Life Technologies), rat anti-HA 3F10 (Roche) and fluorescence-conjugated secondary antibodies (Jackson Laboratories) (1:300). We used biotinylated Peanut Agglutinin to label ring canals (Vector Laboratories)(1:500).
RNA was extracted from hs-bam;bamΔ86 mutant ovaries and made into cDNA using SuperScript II -Strand Kit (Life Technologies), together with a reverse primer for the unique cytoplasmic exon sequence (Supplemental Table 1). We then performed PCR using Rbfox1 specific primers. PCR products were cloned into pENTR (Life Technologies) and swapped into pAHW (Drosophila Gateway Vector Collection) using an LR reaction. Using this approach we isolated clones corresponding to the Rbfox1-RF and Rbfox1-RN (accession number: XXXX) transcripts. HA tagged Rbfox1-RF/RN sequences were cloned into KpnI and XbaI sites of pJFRC28 (Pfeiffer et al., 2012).
α-Tub 3′UTR with nested wild type or control elements were generated by splicing by overlap extension (SOE) PCR and cloned into pCasper-Attb vector containing Venus (Li et al., 2012). Two more rounds of SOE-PCR were used to fuse a segment of the vasa promoter and α-Tub 5′UTR to the reporters. Final PCR products were cloned into pCasper-Attb. To generate PumGCAUG and PumACAUA reporters wild type or mutated forms of pum-RA 3′UTR were synthesized (Integrated DNA Tech.) and cloned into the vasP-α-Tub 5′UTR-Venus reporter mentioned above. For the quantification of, UASt-myrGFP-α-Tub 3′UTR, Rbfox1 and UASt-myrGFP sensor levels, adult Drosophila brains were dissected in AHL, fixed for 20min in 4% paraformaldehyde, washed, mounted and imaged. The obtained images where quantified for mean fluorescence in specific areas of interest.
We used recombineering techniques (Carreira-Rosario et al., 2013) to insert a Zeomycin cassette into a modified version of CH321-94L16 P[acman] clone (Tastan et al., 2010). Specifically the cassette was inserted in the intronic region upstream of the exon that contains portion encoding for F158 and F160 residues. Cassette was homology arms were added to Zeomycin cassette through PCR with Rbfox1-Zeo_F and F158A,F160A_Rbfox1-Zeo_R primers for [FF>AA] or Rbfox1-Zeo_F and F158A,F160A_Ctl_ Rbfox1-Zeo_R for [WT].
To generate the Rbfox1dsRed.1 allele, guide RNAs were designed using http://tools.flycrispr.molbio.wisc.edu/targetFinder and synthesized as 5′-unphosphorylated oligonucleotides, annealed, phosphorylated and ligated into the BbsI sites of pU6-BbsI-chiRNA plasmid (Gratz et al., 2013). Homology arms were PCR amplified and cloned into pHD-dsRed-attP (Gratz et al., 2014)(Addgene). Guide RNAs and the donor vector were co-injected into nosP Cas9 attP embryos at the following concentrations: 250 ng/μl pHD-dsRed-attP donor vector and 20 ng/μl of each of the pU6-BbsI-chiRNA plasmids containing the guide RNAs (Rainbow Transgenics Inc.).
GST-Venus or GST-Rbfox1-RRM recombinant protein was mixed in 10 mM HEPES (pH 7.5), 0.2% Tween-20, 50 mM KCL, 2 mM DTT, 1 μg/μl yeast tRNA, 0.05 μg/μl BSA and 200 U/mL of RNAse inhibitor to a final concentration of 4.5 nM. Non-labeled competitor RNA (1X equals 0.2 ng/ul) was added and incubated for 5 minutes at room temperature. 1X DIG labeled RNA was added to each reaction and incubated for 25 minutes at room temperature. Samples were resolved on a 4% polyacrylamide non-denaturing TBE mini-gel at 4°C. RNA was transferred to a Hybond –N+ membrane, UV-crosslinked and processed according to (Buszczak and Spradling, 2006).
Ovaries were dissected in 1XPBS and cross-linked using 0.08% formaldehyde in 1XPBS for 10 minutes. Fixation was quenched with 2 M glycine. Ovaries placed on ice and rinsed with 1xPBS and then RIPA buffer (50 mM Tris-HCl, 200 mM NaCl, 0.4% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 200 mM NaCl). The samples were lysed in RIPA plus (RIPA, 2.5 mg/ml yeast tRNA, 1 mM PMSF, 50 U/ml Roche RNAse inhibitor) and clarified by centrifugation. For the Rbfox1 sensor mRNA, RIP lysates were mixed with pre-washed Anti-FLAG M2 affinity beads. pumilio RIP lysates were mixed with either Rbfox1 polyclonal antibody or pre-serum, incubated at 4°C for two hou rs followed by addition of protein A agarose beads (Sigma). Samples were then incubated overnight at 4°C under constant mixing. The beads were then washed with 500 μL RIPA plus U (RIPA plus supplemented with 1M Urea) and incubated in 1 mM Tris-HCl (pH 6.8) 5 mM EDTA, 10 mM DTT, 1.0% SDS at 70°C for 45 minutes. Proteinase K was added to a final concentration of 0.1 mg/mL and incubated for 25 minutes at 37°C. RNA was isolated using Trizol and analyzed wi th either One-Step RT-PCR (Qiagen) or by cDNA synthesis followed by qPCR using SYBR Green Master Mix (Applied Biosystems).
We used Poly(A) Tail-Length Assay Kit (Affymetrix) following the instructions provided by the manufacturer.
We would like to thank P. Lasko, Y. Yamashita, the Bloomington Drosophila Stock Center and the Developmental Studies Hybridoma Bank for reagents, Nevine Shalaby and members of the Buszczak and Ramaswami labs for comments and advice, and Jose Cabrera for help in preparing the graphical abstract. A.C.R. was supported by NIGMS (T32GM083831 and then F31GM105332). V.B. was supported by HHMI (Grant#56006776) and NIGMS (T32GM10977601). This work was also supported in various phases by awards from the NIGMS (R01GM086647) and the NIA (R01AG047318) to M.B. and a Science Foundation Ireland Investigator grant award to M.R.
Author ContributionsA.C.R., M.R. and M.B. conceived the project. A.C.R., V.B., J.H., M.R. and M.B. designed and conducted the experiments. A.C.R., V.B., J.H., M.R. and M.B. analyzed the data, wrote and edited the manuscript.
The authors do not declare any conflicts of interest.
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