CNCC-specific deletion of Prmt1 elevates intron retention in the embryonic mandibular process.

(A) Expression levels of PRMT1-9 mRNAs in primary isolated cranial neural crest cells (CNCC) from Wnt1-Cre; RosaTd mouse embryonic heads at E13.5 and E15.5. TPM, transcript per million. (B) Diagram illustrating the isolation of CNCCs from embryonic mandibles, followed by poly(A)+ mRNA isolation and sequencing (n=4 in control and in Prmt1 CKO). (C) Prmt1 deletion in CNCC caused changes in alternative splicing (AS). Changes in AS events were analyzed by rMATS using RNA sequencing data and significant changes in each type of AS were shown in a stacked bar chart. SE, skipped exon. IR, intron retention. MXE, mutually exclusive exons. A5SS, alternative 5’ splice site. A3SS, alternative 3’ splice site. a) The number of intron retention events that increased (Up, yellow color) or decreased (Down, brown color) in Prmt1 deficient CNCCs. (D & E) Intron retention was prevalent in CNCCs and altered by Prmt1 deletion. Track view of genes demonstrating intron retention and further elevated by Prmt1 deletion, illustrated by Pex12, Nkx3-2, Nkiras1, 1810026B05Rik in D or reduced by Prmt1 deletion, illustrated by Tbx1 in E. (F) Quantification of intron expression in Pex12, Nkx3-2, Nkiras1, 1810026B05Rik and Tbx1 by the percentage of intron-retaining mRNAs in each gene, calculated from IRI analysis based on RNA sequencing data. * p<0.05 Prmt1 CKO vs Control. Control: Wnt1-Cre; RosaTd. Prmt1 CKO: Wnt1-Cre; Prmt1fl/fl; RosaTd.

CNCC-specific Prmt1 deletion reduced matrix gene expression in the developing mandibles.

(A) Volcano plot illustrating upregulation of 160 and downregulation of 303 genes in the mandibular primordium of Prmt1-deficient embryos at E13.5, compared to control embryos. (B) Heatmap showing differential gene expression between control and Prmt1-deficient mandibles. The top downregulated matrix genes were labeled. (C) GO analysis of pathway enrichment in downregulated genes demonstrating glycosaminoglycan (GAG) degradation and extracellular matrix (ECM) organization as the top pathways. (D) Ingenuity Pathway Analysis suggesting connective tissue, bone, and cartilage development as affected biological processes based on downregulated genes. Control: Wnt1-Cre; RosaTd (n=4). Prmt1 CKO: Wnt1-Cre; Prmt1fl/fl; RosaTd (n=4).

PRMT1 regulates intron retention in ECM and GAG degradation genes.

(A-C) Intron retention increased in the majority of ECM gene transcripts that were downregulated in Prmt1 deficient embryos, as illustrated by a scatter plot based on intron retention index (IRI) analysis. The red line delineates where intron retention is unchanged. Genes with the top differential IR were represented by red dots and labeled. ECM gene transcripts Bmp7 and Cthrc1 demonstrating higher intron retention in Prmt1- deleted embryos were illustrated by track view in B and quantification of intronic (left) and exonic (right) expression in C. (D) Higher intron retention and lower mRNA level of ECM genes were validated in a panel by RT-PCR. Primers that span the intronic or intron-exon junction region were used to assess intronic expression. Primers that span the exonic region were used to examine exonic expression. (E-G) Intron retention increased in the majority of GAG degradation gene transcripts that were downregulated in Prmt1 deficient embryos compared to control, as indicated by a scatter plot based on IRI analysis. The red line delineates where intron retention is unchanged. Genes were represented by black dots and labeled in red. GAG degradation genes St6galnac3 and Galnt11 demonstrating higher intron retention in Prmt1-deleted embryos were illustrated by (F) track view and (G) quantified for intronic (left) and exonic (right) expression. * p<0.05 Prmt1 CKO vs Control. Control: Wnt1-Cre; RosaTd. Prmt1 CKO: Wnt1-Cre; Prmt1fl/fl; RosaTd.

PRMT1 methylates SFPQ and regulates its expression.

(A) Expression levels of splicing factors SFPQ, SRSF1, EWSR1, TAF15, TRA2B, HnRNPA1, WDR70, G3BP1 in primary isolated CNCCs from Wnt1-Cre; RosaTd mouse embryonic heads at E13.5 and E15.5. TPM, transcript per million. (B-F) SFPQ methylation diminished in the mandibular and maxillary processes of Prmt1 deficient embryos at E13.5. B shows that sagittal sections of mouse tissues were used to analyze the mandibular (area outlined in red, bottom) and maxillary (area outlined in red, top) process. Methyl-SFPQ was detected by proximity ligation assay (PLA) in the embryonic mandible (C) and maxilla (E) of control and Prmt1 deficient embryos. Green puncta indicated methyl-SFPQ, and nuclei were counterstained with DAPI (blue). Representative images are shown for Control (Ca-Cd, Ea-Ed) and Prmt1 CKO (Ce-Ch, Ee-Eh). Higher magnification views in (Cd, Ed) and (Ch, Eh) illustrate methyl-SFPQ (green puncta) in CNCC nuclei, as indicated by white arrows. D&F show quantification of PLA puncta normalized to cell number in four biological replicates, presented as mean ± SEM. (G) SFPQ methylation signal remained robust in the epithelial region of craniofacial structures, while CNCC-specific deletion of Prmt1 caused a drastic reduction in the craniofacial mesenchyme. Higher magnification of the mandibular tip showing mesenchyme and epithelium outlined with dotted lines in Control (Ga-Gc) and Prmt1 CKO (Gd-Gf). (H-K) SFPQ protein expression was significantly reduced in the mandibular and maxillary processes of Prmt1 deficient embryos. The level of SFPQ protein was detected by immunostaining in the embryonic mandible (H) and maxilla (J) of control and Prmt1 deficient embryos, and quantified in I and K. (L-M) SFPQ protein expression declined dramatically in the Prmt1 deficient embryonic head, as detected by Western blotting (L). Quantification was conducted with ImageJ and shown in M. Mx, maxilla. Md, mandible. * p<0.05 Prmt1 CKO vs Control. Control: Wnt1-Cre; RosaTd. Prmt1 CKO: Wnt1-Cre; Prmt1fl/fl; RosaTd.

PRMT1-SFPQ pathway regulates matrix genes in CNCCs.

(A) SFPQ depletion in CNCCs altered intron retention. IR events in genes showing increased (Up, yellow color) or decreased (Down, brown color) intron retention were illustrated by stacked bar graphs. CNCCs were transfected with two independent siRNAs targeting SFPQ, or control siRNA, followed by poly(A)+ mRNA extraction and RNA sequencing. (B) SFPQ depletion in CNCCs altered gene expression. Pie chart demonstrating differentially regulated genes in CNCCs with SFPQ depletion by two independent siRNAs. Shaded areas among the downregulated genes (orange) indicate overlap with IRI up. (C) GO analysis of genes with increased IR events in CNCCs with SFPQ depletion by siRNA#1 (a) or #2 (b). (D) Genes with increased IR events in SFPQ-depleted CNCCs demonstrated 8.28% (64 out of 773) overlap with Prmt1 deficient CNCCs. (E-H) Matrix genes regulated by both Prmt1 deletion and SFPQ depletion. Bar graph showing elevated IR (E, F) and reduced expression (G, H) in both SFPQ-depleted and Prmt1 deletion CNCCs. TPM, transcripts per million. (I-J) Heatmap and GO analysis of SFPQ-regulated genes in CNCC. * p<0.05 siSFPQ vs siControl. siControl, CNCCs transfected with control siRNA. siSFPQ#1 and siSFPQ#2, CNCCs transfected with two independent SFPQ siRNAs. * p<0.05 Prmt1 CKO vs Control. Control, Wnt1-Cre; RosaTd. Prmt1 CKO, Wnt1-Cre; Prmt1fl/fl; RosaTd.

SFPQ regulates intron retention of Wnt signaling and neuronal genes in CNCCs.

SFPQ depletion in CNCCs elevated intron retention and decreased mRNA expression of a group of Wnt signaling components (A) and neuronal genes (B). The levels of mRNA expression (left) and intron retention (right) were illustrated by a two-sided bar graph. TPM, transcripts per million. * p<0.01 siSFPQ vs siControl. siControl, CNCCs transfected with control siRNA. siSFPQ#1 and siSFPQ#2, CNCCs transfected with two independent SFPQ siRNAs.

SFPQ depletion reduces long gene expression via intron retention triggered NMD.

(A) SFPQ binding peaks were mapped to retained intron 1, but not spliced intron 6 of Ptk7. Peak distribution from published Sfpq CLIP-seq data using E13.5 brain (top) and track view of RNA-seq data using siCont or siSFPQ-transfected CNCCs (bottom) for Ptk7, with the retained Ptk7 intron 1 and spliced intron 6 highlighted. (B) SFPQ binding peaks were significantly enriched in retained introns within CNCCs. Violin plot displaying the global distribution of enriched SFPQ binding peaks in introns with elevated retention compared to introns with reduced retention or no change. Peak number was normalized to intron length. P value was indicated at the top. (C) SFPQ binding peaks were preferentially enriched in genes with higher intron retention when compared to genes with no IR change. p = 0.07 using Fisher’s exact test. (E) SFPQ-regulated genes were significantly longer. Violin plot displaying the distribution of length for genes showing increased intron retention (IR Up), decreased intron retention (IR Down), or unchanged intron expression (No Change). (F) SFPQ depletion promoted intron retention and reduced mRNA expression of Col4a2, St6galnac3, and Ptk7 in ST2 cells. Bar chart showing RT-PCR analysis of intronic and exonic expression. * P<0.05 siSFPQ vs. siControl. (G-H) NMD inhibitor NMDI14 induced the accumulation of retained introns and mRNAs of Col4a2, St6galnac3, and Ptk7 in ST2 cells. Bar chart showing RT-PCR analysis of intronic (G) and exonic expression (H) in DMSO or NMDI-treated cells. * P<0.05 NMDI vs. DMSO treatment of the same group.