Fig 2: (A) Through the intersection of the ENCORI and RBPDB databases, it was concluded that hsa_circ_0058493 may interact with YTHDC1. (B) Relative expression of YTHDC1 in 12 pairs of HCC and adjacent tissues was detected by qRT-PCR assays. (C) Top, a point mutation in the binding sequence of hsa_circ_0058493 and YTHDC1. Bottom, the RIP experiment proved that YTHDC1 interacted with hsa_circ_0058493 and IgG antibody was used as a control. (D,E) The expression of YTHDC1 mRNA was detected by qRT-PCR in cells with hsa_circ_0058493 knockdown plasmid or hsa_circ_0058493 overexpression plasmid. (F) Hsa_circ_0058493 was positively correlated with YTHDC1. The P value and correlation coefficient were calculated by Pearson correlation analysis. (G) Nuclear and cytoplasmic separation experiments showed that hsa_circ_0058493 was in the cytoplasm. U6 was used as a positive control in the nucleus while 18S was used as a positive control in the cytoplasm. (H) The knockdown efficiency of the YTHDC1 knockdown plasmid was tested by qRT-PCR. (I) The nucleoplasmic separation experiment showed that after knocking down YTHDC1, hsa_circ_0058493 was transported from the cytoplasm to the nucleus. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.

Fig 3: (A) A CCK-8 assay was used to determine the proliferation of HCC cells after transfection with si-NC + oe-NC, si-YTHDC1 + oe-NC, and si-YTHDC1 + oe-circ. (B) A cloning assay was performed to detected proliferation ability of HCC cells with si-NC + oe-NC, si-YTHDC1 + oe-NC, and si-YTHDC1 + oe-circ. Colony formation rates are shown by histograms. (C) The cell cycle of HCC cells after transfection with si-NC + oe-NC, si-YTHDC1 + oe-NC, and si-YTHDC1 + oe-circ was determined by flow cytometry. As evident in the histogram, more cells arrested in the G1 phase were observed in si-YTHDC1 + oe-NC compared with si-NC + oe-NC. After cotransfection of si-YTHDC1 + oe-circ, the number of cells arrested in G1 phase decreased again compared with si-YTHDC1 + oe-NC. The triangle symbols are for discrimination of G1 vs. S and S vs. G2. (D) The invasion and migration abilities of HCC cells after transfection with si-NC + oe-NC, si-YTHDC1 + oe-NC, and si-YTHDC1 + oe-circ were assessed by Transwell assay. Histograms were used to show the number of invaded and migrated cells. *P < 0.05, **P < 0.01 vs. control group.

Fig 4: Inducible YTHDC1 knockout impairs satellite cell (SC) activation/proliferation.(A) Left: EdU (red) staining of ASC-24 hr and ASC-48 hr from inducible knock out (iKO) and Ctrl mice. Scale bar = 200 µm. Right: quantification of the percentage of EdU+ cells. n = 3 mice per group for ASC-24 hr, n = 4 mice per group for ASC-48 hr. (B) Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO mice and cultured for 48 hr. Scale bar = 100 µm. Right: quantification of the percentage of Pax7+ EdU+ SCs. n = 3 mice per group. (C) Top: immunofluorescence (IF) staining of Pax7 (green) and MyoD (red) of freshly isolated SCs (FISC) (0 hr), ASC-24 hr and ASC-48 hr from Ctrl and iKO mice. Scale bar = 100 µm. Bottom: quantification of the percentage of Pax7+ MyoD+ cells. n = 3 mice per group. (D) Top: IF staining of Pax7 (green) and MyoD (red) on EDL myofibers at 0 hr (freshly isolated), 24 hr, and 48 hr. Scale bar = 100 µm. Bottom: quantification of the number of Pax7+ MyoD+ cells per fiber (n = 4 mice per group for 0 and 24 hr, n = 5 mice per group for 48 hr). (E) Schematic illustration of in vivo EdU assay in Ctrl and iKO mice. (F) Top: EdU staining of the above freshly isolated and fixed SCs at 3 dpi. Scale bar = 200 µm. Bottom: quantification of the percentage of EdU+ cells in iKO vs. Ctrl. n = 3 mice per group. (G, H) Left: RNA-seq was performed in ASC-24 hr or -48 hr from iKO and Ctrl mice. Volcano plot showing the down- and upregulated genes in iKO vs. Ctrl. Right: GO analysis for the downregulated genes. (I) Two independent mAID-YTHDC1 cell lines were treated with DMSO or 5-Ph-IAA (IAA) for the indicated time. EdU assay was performed and the percentage of EdU+ cells was quantified at the designated time points. n = 3 replicates. (J) WT-YTHDC1, or a single (W429A), or a triple (K362A, S363A, and N364A) mutant of YTHDC1 lacking m6A binding ability was transfected with the mAID-YTHDC1 cells then treated with IAA. mAID-YTHDC1 cells treated with DMSO or IAA were used as control. EdU assay was then performed and the percentage of EdU+ cells was quantified. n = 3 replicates. Bars represent mean ± SD for all graphs. Statistical significance was determined using a two-tailed Student’s t-test.

Fig 5: LACE-seq defines transcriptome-wide YTHDC1 binding profiles in myoblasts.(A) Schematic illustration of the experimental design for performing LACE-seq and subsequent combination with MeRIP-seq, bulk RNA-seq, subcellular RNA-seq for defining and elucidating YTHDC1 splicing/export targets and post-transcriptional regulation. (B) LACE-seq was performed in both ASC-48 hr and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. (C) Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. (D) Left: Go analysis for the identified YTHDC1 targets in C2C12. Right: genome tracks for three selected genes. (E) Top: overlapping between the above-identified C2C12 and ASC peaks. Bottom: genome tracks for two selected genes. (F, G) Left: the genome distribution of YTHDC1 binding peaks in C2C12 or ASC. Right: detailed distribution of the YTHDC1 binding peaks on repeat regions. (H) Left: integrating C2C12 MeRIP-seq data with the C2C12 LACE-seq identified 5356 regions (1771 mRNAs, as m6A-YTHDC1 targets). Right: distribution of YTHDC1 binding on the above targets. (I) Left: GO analysis of the above 1771 targets. Right: genomic tracks of a selected target, Phlda3. (J) Left: integrating the C2C12 MeRIP-seq data with the ASC LACE-seq identified 56 regions (50 mRNAs, as m6A-YTHDC1 targets). Right: distribution of YTHDC1 binding on the above-identified target mRNAs. (K) Left: GO analysis of the above 50 targets. Right: genomic tracks of a selected target, Ybx1. (L) C2C12 cells were treated with DMSO or STM2457. m6A modification levels on m6A-YTHDC1 targets were examined by MeRIP-qPCR. Relative enrichment was calculated by comparing the MeRIP/input ratio of DMSO and STM2457. n = 3 biological replicates. (M) C2C12 cells were transfected with pRK5-flag-YTHDC1 and then treated with DMSO or STM2457. YTHDC1 binding to m6A-YTHDC1 targets was examined by RIP-qPCR. Relative enrichment was calculated by comparing the RIP/input ratio of DMSO and STM2457. n = 3 biological replicates. Bars represent mean ± SD for all graphs. Statistical significance was determined using a two-tailed Student’s t-test.