Low‐density lipoprotein receptor‐related protein 6 regulates alternative pre‐mRNA splicing

Abstract Low‐density lipoprotein receptor‐related protein 6 (LRP6) serves as a Wnt coreceptor. Although Wnt/LRP6 signalling is best known for the β‐catenin‐dependent regulation of target genes in tissue development and homeostasis, emerging evidence demonstrates the biological aspects of LRP6 beyond a Wnt coreceptor. Whether LRP6 modulates tissue development in a Wnt/β‐catenin signalling‐independent manner remains unknown. Using a model of striated muscle development, we observed that LRP6 was almost undetectable in proliferating myoblasts, whereas its expression gradually increased in the nucleus of myodifferentiating cells. During myodifferentiation, LRP6 modulated the muscle‐specific splicing of integrin‐β1D and consequent myotube maturation independently of the β‐catenin‐dependent Wnt signalling. Furthermore, we identified that the carboxy‐terminal serine‐rich region in LRP6 bond to the adenine‐rich sequence within alternative exon D (AED) of integrin‐β1 pre‐mRNA, and therefore, elicited AED inclusion when the spliceosome was recruited to the splice site. The interaction of LRP6 with the adenine‐rich sequence was sufficient to overcome AED exclusion by a splicing repressor, polypyrimidine tract binding protein‐1. Besides the integrin‐β1, deep RNA sequencing in different types of cells revealed that the LRP6‐mediated splicing regulation was widespread. Thus, our findings implicate LRP6 as a potential regulator for alternative pre‐mRNA splicing.

extensive organ defects and neonatal lethality, which resembles the phenotypes caused by mutations in individual Wnt genes. 4,5 Moreover, evidence for the pathophysiological relevance of Lrp6 mutation and deficiency, such as Alzheimer disease, coronary artery disease and cardiac ischemia, have emerged, and presumably the aberrant Wnt signalling mediates the functional link. 2,6 The biological aspects of LRP6 beyond a Wnt coreceptor have been gradually recognized. Our recent study demonstrated a scaffolding role of LRP6 in the membrane targeting of connexin 43 and thus gap junction assembly in the mouse heart. 7 Wan et al reported the property of LRP6 as a trafficking adaptor, showing that Lrp6 knockdown disrupts the localization of Gas to the plasma membrane and impairs a functional G protein-coupled receptor (GPCR) signalling pathway for the production of cAMP. 8 Interestingly, several lines of evidence also documented that a soluble LRP6 intracellular domain can translocate to the nucleus and directly modulates the activity of the Wnt responsive transcription factor TCF/LEF-1, 9,10 suggesting the potential transcriptional and post-transcriptional activities of LRP6 protein. These reports led us to suspect whether LRP6 modulates tissue development and homeostasis in a Wnt/b-catenin signalling-independent manner, and if so, to delineate the molecular mechanisms.
In the present study, using a well-established model of striated muscle development, 11
The medium was changed every 24 hours before the test. To obtain the knockdown of Lrp6 in C2C12 cells, adenovirusmediated Lrp6 shRNA that targets the sequence GCACTACAT-TAGTTCCAAA and control shRNA (ATAGCTACAATCGCAATCT) were transfected into cells. To quantify the myodifferentiation, we calculated the fusion index as the average number of nuclei in MHC-positive cells with at least three nuclei above total number of nuclei, and the number of nuclei per myotube was measured using the NIH Image J software.

| Isolation and culture of neonatal rat cardiomyocytes
The isolation and culture of neonatal rat cardiomyocytes (NRCMs) were performed according to the procedures previously described. 7

| Cellular fractionation
Cytoplasmic and total nuclear proteins were extracted using the Cel-LyticTM NuCLEARTM Extraction Kit (NXTRACT, Sigma) according to the manufacturer's instructions.

| Immunofluorescence microscopy
HeLa cells were transfected with vectors expressing the flag-fused protein containing the full-length LRP6 for 48 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 10 minutes, After nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, 5 lg/mL), the cells were visualized using a Leica confocal laser-scanning microscope.

| mRNA stability assays
Itgb1D mRNAs in NRCMs were quantified relative to 18S rRNA at various times after addition of actinomycin D (ActD, 6.5 lg/mL, Sigma-Aldrich) to the culture medium. Relative quantification values at 0 hour were set to 1.

| Protein purification
To obtain the purified proteins of the full-length LRP6, the vector expressing LRP6 was constructed. The corresponding sequence was amplified from Lrp6 plasmid (Origene) by PCR and was subcloned into the pcDNA3.1 expression vector (Invitrogen) using BamH1 and Xbal restriction sites. The DNA sequence of the flag tag was attached to the C-terminus of Lrp6. The plasmid was transfected into the 293T cells for 48 hours using Lipofectamine3000 (Invitrogen), and the overexpressed LRP6 proteins were purified using FLAGâ M Purification Kit (Sigma).

| Biotinylated RNA pull-down assays
The full-length U1-U6 snRNAs (rat) were transcribed in vitro using T7 RNA polymerase (ThermoFisher), and then were labelled using Pierce RNA 3 0 end desthiobiotinylation kit (ThermoFisher). 150 pM RNA probes were incubated with 50 lL streptavidin magnetic beads (Sigma) at room temperature for 25 minutes. Then, 400 ng of the purified flag-tagged LRP6 fusion proteins were incubated with RNAbeads complex at 4°C for 50 minutes. The proteins bound to U1-U6 snRNAs were stripped by elution buffer and analysed by Western blotting with anti-LRP6 antibody (C5C7, CST).

| Plasmid construction
To construct recombinant vectors of U1-U6 snRNAs, their sequences were amplified from rat cDNAs and subcloned into the pCMV6-entry vector (Origene) using SgfI and XhoI restriction sites.  Table S1.

| Minigene constructs and site-directed oligonucleotide mutation
The minigene constructs were assembled in the pCMV6-entry vector  Table S1.

| Luciferase assays
To measure the transcription activity of Itgb1 gene, pGL3-Itgb1 promoter luciferase reporter vector and pRL-TK (Promega) were cotransfected in the HeLa cells, which were subject to Lrp6 silencing by the targeted siRNAs for 48 hours. After another 24 hours, cells were harvested and analysed using a Dual-Luciferase reporter assay kit (Promega).

| Immunoblotting analysis
Proteins were extracted on ice using a RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor-cocktail (Roche).  Twenty-four hours later, Itgb1-minigene reporters were overexpressed for another 24 hours in these cells with Lipofectamine 3000 (Invitrogen). Cellular RNAs were extracted using TRI reagent (Ambion) and then treated with RNase-free DNase I (Promega) for 30 minutes at 37°C. Complementary DNA was generated by reverse transcription with a poly T primer for 30 minutes at 37°C using reverse transcriptase (Takara). Semi-quantitative RT-PCR was performed through GoTaqâ Green Master Mix (promega) on a PCR system (Applied Biosystems), and the products were identified by agarose gel electrophoresis.

| Statistical analysis
All data are presented as the means AE standard errors of the mean (SEM). The data shown were the averages of at least three biological replicates. No inclusion/exclusion criteria were used. Statistical analyses were performed using Prism Software (GraphPad). The statistical significance of the difference between two sets of data was assessed using an unpaired, two-tailed Student's t test and one-way ANOVA with Bonferroni's post-hoc test. A P value less than .05 was considered to be significant.

| LRP6-mediated muscle development couples to the muscle-specific splicing of Itgb1D
To test whether LRP6 modulates tissue development and homeostasis in a Wnt signalling-independent manner, we utilized a C2C12 myoblast model that recapitulates important features of striated muscle development. These myoblasts underwent a program of proliferation and myodifferentiation to form multinucleated myotubes ( Figure 1A,B). During the proliferative phase, LRP6 protein was barely detectable in C2C12 myoblasts. Its expression prominently increased when myoblasts entered into myodifferentiation, and peaked when myotube formed ( Figure 1C). The coincidence of the onset of LRP6 expression with the timing of myoblast withdrawal from the cell cycle implied a major function for LRP6 in commitment of differentiating muscle cells, which was evidenced by the findings that LRP6 deficiency greatly retarded, whereas its overexpression promoted, the myotube fusion ( Figure 1D).
During striated muscle development, integrin-dependent cellmatrix adhesion sets the starting point for myofibrillogenesis. 12 Different splicing isoforms of Itgb1 are differentially expressed in foetal (Itgb1A) and mature muscle cells (Itgb1D), and the latter is essential for myotube fusion. 13,14 We found that ITGB1D protein was gradually increased in parallel with LRP6 protein during myodifferentiation ( Figure 1E). LRP6 deficiency down-regulated the expression of ITGB1D protein, but did not affect the total ITGB1 proteins (Figure 1E). The reduction of ITGB1D proteins was not involved in the transcriptional change of Itgb1 and the degradation of Itgb1D mRNA and protein ( Figure S1 and S2). However, while the overall amount of Itgb1 mRNAs appeared normal, the transcript levels of Itgb1D were decreased and concurrently the expression of Itgb1A mRNAs up-regulated by LRP6 deficiency, suggesting the deregulated splicing switching of Itgb1A to muscle-specific Itgb1D isoforms ( Figure 1F).
These evidence indicate that LRP6 may act as a trans-acting regulator mediating the functional splicing of Itgb1 pre-mRNAs.

| LRP6 interacts with the core spliceosome components to direct muscle-specific splicing of Itgb1D in the nucleus of striated muscle cells
As the splicing regulation is well characterized in the eukaryotic nucleus, 15 we expected that LRP6 is physiologically present at appreciable levels in the nucleus. Unexpectedly, LRP6 protein was almost undetectable in the nucleus of C2C12 myoblasts. However, as soon as myodifferentiation commenced, the nuclear expression of LRP6 was dramatically enhanced (Figure 2A), which was positively pertinent to the degree of myodifferentiation. Interestingly, the nuclear expression of LRP6 protein was constantly detected in some Nuclear pre-mRNA splicing is carried out in the spliceosome, a dynamic RNA-protein complex composed of five small nuclear RNAs (U1/2/4/5/6 snRNAs) together with associated splice factors. 16 At the initial step of splicing, U1 and the U2 auxiliary factors (U2AF) bind to the 5 0 and 3 0 splice site, respectively. Successively, U2 tethers to the branch point, and a preformed complex of U4/5/6 tri-snRNPs is recruited to the intron. After release of the U1 and U4, the splicing reaction is catalysed. Using RNA-immunoprecipitation (RIP) and in vitro biotinylated RNA pull-down assays, we identified that LRP6 bond to U4/5/6 snRNAs ( Figure 2B). In addition, the immunofluorescent staining also revealed the partial co-localization of endogenous LRP6 protein with the splice factors U2AF65, C1 and PTBP1 in the nucleus of HeLa cells ( Figure 2C). The U2AF65 protein contains a serine/arginine (SR)-rich domain that recruits itself to  17,18 These results suggest that LRP6 may function at the catalytic step of splicing.
Moreover, the specific activity of LRP6 on Itgb1 pre-mRNA was confirmed using a splicing reporter minigene in which exclusion of the alternative exon D (AED) contributes to Itgb1's elasticity (Figure 2D). Considering the case that the efficacy of the minigene expression was low in C2C12 cells, we conducted the activity analysis of the splicing reporter in intact cultured neonatal rat cardiomyocytes that share the same expression profiles of Itgb1A/1D isoforms. Reduction of LRP6 induced the exclusion of AED in neonatal rat cardiomyocytes; however, LRP6 protein did not affect the usage of AED in HeLa cells, indicating that LRP6-mediated Itgb1 splicing is muscle-specific and additional splicing cofactors missing in non-muscle cells are necessary for the splicing process.

| The C-terminal serine-rich region in LRP6 directly targets the adenine-rich sequence within AED to determine the usage of AED
The exclusion or inclusion of alternative exons highly depends on the position and context of splicing cis-regulatory sequences within alternative exons or the flanking introns, including exonic splicing enhancers or silencers (ESEs or ESSs) and intronic splicing enhancers or silencers (ISEs or ISSs) that recruit splicing regulators. 16,17 Using the RIP approach, we observed that both mature Itgb1D mRNA and Itgb1 pre-mRNA, which share the 81-bp AED sequence, were detected in LRP6 protein-immunoprecipitated transcripts ( Figure 3A), implicating that the AED was the potential cis-regulatory element.  Figure 3D), which was identical to the effects by 20-bp deletion, whereas the mutation of C to G between 51 and 60 bp partially weakened the AED inclusion, implicating the adenine-rich sequence within AED as the core ESE.
Next, we defined the amino acid resides responsible for targeting the ESE in AED. The web-based RNA-binding analysis showed that the 73-132 amino acid regions within LRP6-ICD domain are highly preferential for RNA binding ( Figure 3E), in which the serine is rich.
Using the site-directed mutagenesis, we identified that the nuclear leading sequence (NLS)-mediated expression of the ICD mutant containing a Ser-to-Ala substitution (NLS-ICDMut) induced the exclusion of AED in the neonatal cardiomyocytes expressing wild-type minigene ( Figure 3E), indicating that the serine residue was critical for the RNA splicing activity. Overall, LRP6 may act primarily as a splicing regulator to facilitate the inclusion of AED within Itgb1 pre-mRNA in striated muscle cells.

| LRP6-mediated AED use is sufficient to overcome exon skipping by splicing repressors
The usage of a particular exon is usually subject to antagonistic factors-mediated bidirectional regulation. 17 Members of the hnRNP family often cause exon exclusion through binding to splicing cis-regulatory elements. 18 During myodifferentiation, the hnRNP A1 (also known as hnRNP I) and PTBP1 exhibited the expression pattern opposite to LRP6 protein ( Figure 4A). Examination of the splicing minigene revealed the presence of binding sites for hnRNP A1 and PTBP1 in the flanking introns of AED ( Figure 4B). Reduction of hnRNP A1 did not affect the inclusion of AED in neonatal cardiomyocytes ( Figure 4C), and LRP6 did not affect the protein expression of hnRNP A1 and PTBP1 ( Figure 4D). Although PTBP1 reduction promoted the inclusion of AED, the LRP6 deficiency-induced AED exclusion was greatly counteracted by PTBP1 down-regulation (Figure 4E,F), indicating that targeting of LRP6 to ESE within AED was sufficient to overcome the AED exclusion by PTBP1 splicing repressor ( Figure 4G), achieving the splicing of Itgb1D isoform.

| LRP6 guides the global regulation of AS
To determine whether the LRP6-mediated splicing regulation is a more widespread phenomenon, we performed deep RNA sequencing of cells from rat and human. We detected a substantial number of Lrp6 loss-induced alternative splicing events, and up to nearly 50% of them were exon-skipped ( Figure 5A-C). This LRP6-dependent gene network includes Itgb1, whose alternative splicing was expected based on the above experiments and thus validated our approach. A gene ontology analysis of the conserved LRP6-driven network showed enrichment for 94 genes encoding proteins mainly involved in cellular and metabolic processes ( Figure 5D). Among them, a decade of genes with LRP6-dependent isoform expression showed identical splicing patterns on the exon level across species (Table S2). Moreover, we identified that among the 94 genes that are regulated by LRP6 in the three types of cells, only two genes are involved in the Wnt/b-catenin signalling pathway, and 92 genes are not associated with this signalling (Table S2). The RNA sequencing results were verified for several additional genes other than Itgb1 ( Figure 5E). All of these data supported the notion that LRP6 guides the global regulation of alternative splicing.  19 The process requires multiple interactions between pre-mRNAs, small nuclear ribonucleoproteins and splicing factors. Regulation of this process is highly complicated, depending on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions.

| DISCUSSION
Deciphering the splicing code requires understanding of splicing regulatory RNA-binding proteins (RBPs) and their cis-acting binding sites. 16 There are virtually no data on the sequence preferences of RBPs in most organisms. Predictions of splicing regulatory RBPs from sequence features and tissue splicing data have been limited by the fact that the same genomic sequences are recognized differently by a given RBP in different cell types, leading to only a fraction of RBPs studied. 20 Thus, the unexpected finding of the splicing regulatory RNA-binding landscape of LRP6 may extend our understanding of alternative splicing regulation.
The interpretation of the regulation of RBPs on a given RNA target exceedingly depends on the cell type. This scenario is clearly observed in the splicing regulation of LRP6 on Itgb1D, which occurred in striated muscle cells including myodifferentiating myoblasts and cardiomyocytes. In addition, splicing is highly variable as mRNA-RBP interactions are transient and of relatively low specificity. Regulatory RBPs must function in conjunction with specific components of the core splicing machinery to guide splice site selection and pairing. Classic trans-acting splicing regulators are SR proteins (characteristic arginine-/serine-rich domains at the C-terminal end of SR proteins) and hnRNPs. 17 In general, these SR proteins have a binding preference for purine-rich exonic sequences. 17 Interestingly, we identified the interactions of LRP6 with the core spliceosomal components (Figure 2). Taking advantage of site-directed mutagenesis, we also uncovered the RNA-recognition motif Pre-mRNA splicing is conditionally regulated by signal transduction pathways. 16 Although LRP6 protein has long been thought to mediate the Wnt/b-catenin signalling, our recent findings indicated that LRP6 is not necessary for transducing Wnt signalling in striated muscle cells. 7 Especially, despite the soluble LRP6-ICD has the potential to modulate the activity of b-catenin-coupled transcription factor TCF/LEF-1, affecting the transcription of target genes, 9 we did not observe the effect of LRP6 on Itgb1 transcription ( Figure 1 and Figure S1). Instead, the downstream effector b-catenin of the Wnt signalling demonstrated a transcriptional regulation on Itgb1D production ( Figure S3). Additionally, LRP6 had no effects on the expression of b-catenin proteins in myocytes. Thus, in the case the splicing of Itgb1D, LRP6 works in the Wnt/b-catenin signalling-independent manner. However, as far as the control of Itgb1D expression is concerned, LRP6 may exert an effect antagonistic to the Wnt/b-catenin signalling.
In sum, our findings identify LRP6 as a potential splicing regulator. The RNA-binding landscape of LRP6 may also provide insights into the post-transcriptional RNA processing, given that RBPs can modulate diverse post-transcriptional processes including mRNA transport, localization, stability and translation, and microRNA inhibition. 21 In addition, the disease-relevant mutations of Lrp6 have been reported in its splicing regulatory C-terminus, 2 and thus there remain Functional categories of a conserved set of 94 genes with LRP6-dependent AS between humans and rats. (E) The AS of genes identified by RNA-seq was verified through RT-PCR analysis in NRCMs. WT, wild type; KD, Lrp6 knockdown great interests to identify whether and how LRP6 affect the potential splicing events linked to tissue and organ pathophysiology.