Smith JA , Curry EG , Blue RE , Roden C , Dundon SER , Rodríguez-Vargas A , Jordan DC , Chen X , Lyons SM , Crutchley J , Anderson P , Horb ME , Gladfelter AS , Giudice J .

T32 CA009156 NCI NIH HHS , R01 HD084409 NICHD NIH HHS , R01 GM130866 NIGMS NIH HHS , UL1 TR002489 NCATS NIH HHS , R25 GM089569 NIGMS NIH HHS , P30 DK056350 NIDDK NIH HHS , K99 GM124458 NIGMS NIH HHS , R01 GM081506 NIGMS NIH HHS , P40 OD010997 NIH HHS , R35 GM126901 NIGMS NIH HHS , R25 GM055336 NIGMS NIH HHS , F32 GM136164 NIGMS NIH HHS , Howard Hughes Medical Institute , R00 GM124458 NIGMS NIH HHS , P30 CA016086 NCI NIH HHS

Xla.Tg(actc1:GFP){Mohun} + fxr1 CRISPR (Fig.1.F)
Xla.Tg(actc1:GFP){Mohun} + fxr1 CRISPR (Fig.1.G)
Xla.Tg(actc1:GFP){Mohun} + fxr1 MO (Fig.1.E)
Xla.Tg(actc1:GFP){Mohun} + fxr1 MO (Fig.1.F,G)
Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP){Grngr} + fxr1 MO (Fig.1.C,D)

	Graphical Abstract
	Figure 1. Splicing of fxr1 exon 15 regulates Xenopus development. (A) Conservation of FXR1 exon 15-aa sequences in human (Homo sapiens), mouse (Mus musculus), X. laevis, X. tropicalis, and zebrafish (Danio rerio). Red indicates deviation relative to mouse/human sequence. Mouse exon 15 is underlined. (B) A MO targeting exon 15 (mo-e15i15) was used in X. tropicalis. Successful blocking of exon 15 inclusion was confirmed by RT-PCR. (C) Representative images of uninjected embryos or those injected with a mo-control or the mo-e15i15 (X. tropicalis) at stage 45. (D) X. tropicalis embryos at stage 45. RFP signal marks the somites of uninjected, mo-control, or mo-e15i15–treated embryos. (E) Position of mo-i14e15 and sgRNAs within the X. laevis genome. Representative images and quantification of normal or two abnormal phenotypes (1 and 2) for uninjected, mo-control, mo-i14e15, or sgRNA-treated embryos. (F and G) X. laevis embryos at stages 35–38. GFP signal marks the somites of uninjected, injected (mo-control or mo-i14e15), and Cas9 protein injected with sgRNAs targeting exon 15 or introns 14 and 15 for both 5S and 5L. White rectangles indicate the region of the somites magnified in the insets in (F). All scale bars, 1 mm. Fig. 1, E and F, is linked to Fig. S1.
	Figure S1. Sequences of fxr1 exon 15 from sgRNA-treated embryos. Sequences of fxr1.S and fxr1.L from five randomly selected X. laevis embryos injected with the sgRNA T1 (green) and T3 (underlined). sgRNA-injected embryos had deletions (dashed), uncallable nucleotides (N), and mismatches (red) compared with reference sequences. Deletions roughly correspond to 3–4 nt upstream of the protospacer adjacent motif (PAM) sequences (magenta). Fig. S1 is linked to Fig. 1, E and F.
	Figure 2. Alternative splicing regulates FXR1 in a tissue- and developmental stage–specific manner. (A) Mouse Fxr1 gene structure. The arrow indicates transcription initiation site. Numbers indicate the exons. Alternatively spliced exons are indicated in colors. (B) Location of the alternatively spliced region within FXR1. Darker purple in isoforms A and B denotes different amino acids (AA). C, C-terminus; N, N-terminus; NES, nuclear export signal; NLS, nuclear localization signal. Blue, yellow, red, and pink colors correspond to the alternatively spliced exons shown in A. IDD is colored in purple, the RGG in green, and the KH domains in orange. (C and D) Splicing of exons 15 and 16 of Fxr1 pre-mRNA was evaluated by RT-PCR in nonstriated muscles of neonatal (P4.5) and adult (4-mo-old) mice (C). The PSI of exon 15 or exon 16 and the abundance of transcripts evaluated by densitometry (D). (E) Fxr1 pre-mRNA splicing was similarly evaluated in mouse skeletal muscles (sk muscle) and hearts at embryonic day 15.5 (E15.5), P4.5, and adulthood. (F) FXR1 pre-mRNA splicing was similarly evaluated in commercial human RNA samples. Data are means ± SEM, *P ≤ 0.05 (one-way ANOVA test with Bonferroni correction for multiple comparisons), n = 3–4 (mouse tissues), n = 2 (fetal human tissues), n = 3 (adult human tissues). Fig. 2 is linked to Fig. S2.
	Figure S2. Alternative splicing events in exons 12 and 13 of Fxr1 pre-mRNA and expression of FXR1 protein isoforms during tissue development. (A) Scheme of part of Fxr1 gene showing the alternative splicing events in exons 12 (insert of 87 nt) and exon 13 (alternative splice site leading to the inclusion or skipping of a 78-nt region). To evaluate the splicing patterns by RT-PCR, the primers designed to target the constitutive flanking exons were Fxr1-87-78-f (forward) and Fxr1-87-78-r (reverse). (B) Different tissues were collected from neonatal (P4.5) and adult (4-mo-old) mice. Alternative splicing was evaluated by RT-PCR and quantified by densitometry. n = 3–4 (P4.5 tissues) samples from two to nine pooled neonates each depending on tissue type, n = 4 (adult tissues) animals. (C) Adult mouse tissues were evaluated by Western blots using a monoclonal antibody against the N terminus of FXR1 protein that thus recognizes all of the splice variants. n = 2 animals. Data are means ± SEM, *P ≤ 0.05 (one-way ANOVA test with Bonferroni correction for multiple comparisons). sk muscle, skeletal muscle. Fig. S2 is linked to Fig. 2.
	Figure 3. C2C12 cell differentiation reproduces Fxr1 splicing transitions. RNA or protein was extracted at undifferentiated stage (undiff) and differentiating (D0 to D8) C2C12 cells. (A) Splicing of exons 15 and 16 was evaluated by RT-PCRs. The PSI was determined by densitometry. (B) Inclusion or skipping of the 87-nt insert in exon 12 of Fxr1 pre-mRNA. (C) Protein lysates were analyzed by Western blotting. Data are means ± SEM, *P ≤ 0.05 versus undifferentiated stage (one-way ANOVA test with Bonferroni correction), n = 3–5 experiments. Fig. 3 is linked to Fig. S3, A–E.
	Figure 5. FXR1 phase separation is concentration dependent. (A) Whole-cell extracts from WT U2OS cells or U2OSΔFFF cells transiently transfected with either an empty vector (pAGB1139) or PCMV2xTetO2-mNeonGreen-FXR1 isoform E (pAGB1162) were prepared. Extracts were analyzed by Western blot. Right: Quantification of protein levels normalized to a loading control. (B–G) U2OΔFFF cells were transiently transfected with an empty vector (pAGB1139), PCMV2xTetO2-mNeonGreen-FXR1 isoform A (pAGB1189) or isoform E (pAGB1162). (B) Representative images of cells at increasing Tet concentrations. Scale bar, 5 µm. (C) Quantification of droplet formation for individual cells. n > 720 cells from three independent experiments. (D) Zoomed-in images showing examples of irregular droplets, small spheres, and large spheres. Scale bar, 5 µm. (E) Quantification of droplet morphology. n > 185 cells from three experiments. (F–G) The cell lysate and pellet were analyzed by Western blot as described in A. The percentage of protein in the pellet was quantified as the amount in the pellet out of the amount in the whole-cell extract. Data are means ± SEM. Supe, supernatant; WCE, whole-cell extract.
	Figure 6. RNA promotes and phosphorylation inhibits FXR1 association in vitro. (A) Representative images of FXR1 isoforms A and E aggregates (2 or 4 µM) in the presence or absence of luciferase RNA (magenta). Scale bars, 10 µm. (B) Predicted disorder (IUPRED), unspecified kinase score and CK2 kinase scores for serine are marked for each aa. (C) Representative images of isoform A and E aggregates with or without RNA and with or without phosphorylation (Phos). Scale bars, 10 µm. (D–G) Quantification of particle area (D), RNA fluorescence within particles (E), number of particles (F), and number of particles >0.3 µm2 using the images represented in C. ***P < 0.001, **P < 0.01, *P < 0.05 (Student’s t test, two sided). ns, not significant.
	Figure S4. RNA addition and protein phosphorylation influence FXR1 aggregation in vitro. (A) Luciferase RNA accelerates isoform E aggregation. Representative images for either 4 or 2 µM isoform E with or without luciferase RNA after 3, 4, 5, 6, and 24 h. Scale bars, 10 µm. (B) FMR1 Predicted disorder (IUPRED) and unspecified kinase score for serine are marked for each aa. Predicted phosphorylated serines are uniformly distributed across the protein. (C) Coomassie staining of the in vitro–purified FXR1 isoforms A and E. Phosphorylation (Phos) results in a band shift with respect to the control (Ctrl) or mock-treated samples. (D) Representative images of 2 µM FXR1 isoform E (shown in white) with or without RNA and with or without phosphorylation (Phos) at 3, 4, 5, 6, and 24 h. Scale bars, 10 µm. Fig. S4 is linked to Fig. 6.
	Figure 7. Phase separation is RNA binding dependent. (A) Location of mutations in the domains of isoform E. (B and C) U2OSΔFFF cells were transfected with WT PCMV2xTetO2-mNeonGreen-FXR1 isoform E (pAGB1162), plasmids with mutated KH1 (pAGB1171), KH2 (pAGB1172), KH1/2 (pAGB1173), RGG (pAGB1174), RGG and KH1/2 (pAGB1175), complete IDD (pAGB1176), or partial IDD (pAGB1177). All constructs were induced with 25 ng/ml Tet to endogenous levels. Black Xs indicate mutated regions. (B) Quantification of droplet formation. n > 350 cells from three experiments. (C) Representative images of cells expressing the different mutants. Scale bar, 5 µm. (D) Whole-cell extracts (WCE), lysate, and pellets were analyzed by Western blotting using an antibody against FXR1. Right: percent protein in the pellet quantified as in Fig. 5. (E) Quantification of droplet morphology using the same three categories that were used in Fig. 5. Data are means ± SEM.

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