Fig 2: Mov10 is required for neurite outgrowth and neuronal morphology. a–c Brightfield images of N2a (WT), Mov10 knockout N2a (KO), and Mov10 transgene rescue of KO (Rescue). Scale bar represents 200 μm. d Quantification of neurite length of WT, KO, and Rescue, analyzed by one-way analysis of variance (ANOVA) (F (2,19) = 32, p = 0.000, p values, ** 0.01, *** 1.60484E-06). Error bars represent SEM. There were 500–800 differentiated neurons counted from triplicate experiments, and a total of 11 images were counted per condition. Lower panel is Mov10 immunoblot of WT, KO, and Rescue; eIF5α is loading control. e Schematic of significantly changed genes between WT undifferentiated and differentiated N2a. The number of differentially expressed genes as determined by Cuffdiff (p value <0.005 and fragments per kilobase million, FPKM >1) under both conditions is displayed (top). Venn diagram of genes differentially expressed in the KO versus WT. Orange (813): genes identified from comparison between undifferentiated WT and KO; green (781): genes identified from comparison between differentiated WT and KO; purple (513): Mov10-regulated genes (bottom). f Enrichment map of top Gene Ontology terms for the 513 Mov10-regulated genes (DAVID, p value <0.025) showing enrichment for genes related to nervous system development, axon guidance, and neuron projection. Fraction of genes up- (blue) and down- (orange) regulated in KO cells. g Microtubule-associated protein 1b (Map1b) immunoblot from WT, Mov10 KO, and rescue. Gapdh is the loading control. h Significantly changed genes between WT differentiated and undifferentiated (324). Of those genes, 64 significantly changed in the opposite direction of WT in the KO

Fig 3: Mov10 binds mRNAs encoding proteins involved in neuron projection and cytoskeleton by RIP, iCLIP. a GO analysis of RIP mRNAs from postnatal brain. Y axis: GO terms for Cellular Compartment; X axis: negative log (base 10) of the ten lowest p values (see Additional file 5). b GO analysis of iCLIP mRNAs from postnatal brain. Y axis: GO terms for Cellular Compartment and Molecular Function; X axis: negative log (base 10) of the p values, showing only terms with p values of 10–6 or lower. Right: Autoradiograph of Irr and Mov10 IP from P0/P1 brains from iCLIP (see Additional file 7)

Fig 4: RGG box of FMRP attenuates AGO association and subsequent degradation of MOV10-FMRP co-bound mRNAs. (A) (Left) Immunoblot (ib) of endogenous proteins encoded by mRNAs co-bound by FMRP–MOV10 in HEK293 cells expressing FMRP or FMRP-ΔRGG. eIF5B is the loading control. Vertical line denotes the proteins that are ‘protected’. (Right) Quantification of immunoblots (n = 3), error bars represent SD, *P < 0.05, ** P < 0.001 Student t-test. (B) RT-qPCR of IP’ed AGO- associated mRNAs in cells expressing WT FMRP or FMRP-ΔRGG, (n = 3), error bars represent SD, ** P < 0.001 Student t-test. Statistical significance is for all four protected RNAs, MAZ through HN1L. (C) (Top) AGO was IP’ed and examined for GFP-tagged FMRP and FMRP-ΔRGG (GFP). (Bottom) GFP tagged FMRP and FMRP-ΔRGG were IP’ed and examined for AGO. (D) Luciferase activity of the MAZ 3′ UTR in cells expressing the WT FMRP or FMRP-ΔRGG constructs, (n = 3), error bars represent SD, ** P < 0.001 Student t-test. All experiments were performed in triplicate, representative immunoblots are shown.

Fig 5: FMRP binds the N-terminus of MOV10 through its KH1 domain and to AGO through its N-terminus in a phosphorylation dependent manner. (A) Integrated Genomics Viewer (IGV) screen shot of the 3′UTRs (stop codons indicated) of two protected mRNAs, MAZ and HN1L. CLIP regions in which FMRP, MOV10 and AGO2 binding are coincident with rG4s are shown in red boxes, indicated by nucleotide position at the top. rG4s, determined by rG4-seq, are troughs (indicated by red arrows). (B) A GFP-tagged FMRP construct was co-transfected with an HA-tagged N-terminal MOV10 (NMOV10-HA) or HA-tagged C-terminal MOV10 (CMOV10-HA) construct into HEK293 and immunoprecipitated (IP’ed) with an anti-eGFP antibody and examined by immunoblot (ib): antibody indicated on the left. IgG indicates the IP’ing antibody alone. RNase indicates treatment (+) or not (−) with RNase A. (C) (Top) eGFP-tagged KH1 domain of FMRP was IP’ed and examined for mCherry tagged N-terminal MOV10 (mCher). (Bottom) eGFP-tagged FMRP WT or ΔKH1 was IP’ed with anti-eGFP and examined for association with N-terminal MOV10 (mCher), three biological replicates are shown, (n = 3). (D) N-terminal half of FMRP (N-term through KH2 domain) or C-terminal half of FMRP (KH1 through C-terminus) was IP’ed and AGO association examined with the pan-AGO antibody 2A8, which recognizes the four AGO family members in the presence or absence of RNase A. (E) The primary phosphorylation site of murine FMRP (S499) was substituted to alanine (Ala) or aspartic acid (Asp) and association with AGO examined by eGFP-FMRP-IP. AGO co-IP was quantified by normalizing to AGO and transgene protein levels, (n = 3), error bars represent SD, *P < 0.05, Student t-test. (F) Based on their respective interacting domains, a representation of a dynamic RNP complex involving FMRP, MOV10 and AGO proteins in the 3′ UTR of an mRNA (stop codons indicated) bearing an RNA G-Quadruplex (rG4) with an MRE site (red). N denotes N-terminus. All experiments were performed in triplicate, representative immunoblots are shown.