1. Nuclear m6A Reader YTHDC1 Regulates mRNA Splicing
Wen Xiao, Samir Adhikari, Ujwal Dahal, Yu-Sheng Chen, Ya-Juan Hao, Bao-Fa Sun, Hui-Ying Sun, Ang Li, Xiao-Li Ping, Wei-Yi Lai, Xing Wang, Hai-Li Ma, Chun-Min Huang, Ying Yang, Niu Huang, Gui-Bin Jiang, Hai-Lin Wang, Qi Zhou, Xiu-Jie Wang, Yong-Liang Zhao, Yun-Gui Yang 
Molecular Cell 
Volume 61, Issue 4, Pages (February 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 61, 507-519DOI: (10.1016/j.molcel.2016.01.012)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
YTHDC1, SRSF3, and SRSF10 Are Spatially Related with Their Binding Regions and Differentially Regulate AS Events
(A) Enrichment of YTHDC1 binding clusters in longer exons. The length distribution of the exons containing YTHDC1 binding clusters (yellow) showed a longer size compared with that of all exons in the human genome (green).
(B–D) Boxplot showing PSI-level changes of upstream, target, and downstream exons (left, middle, and right) in YTHDC1- (B), SRSF3- (C), and SRSF10 (D)-deficient HeLa cells compared with control. Red and green boxes represent the increased inclusion or skipping level of exons, respectively, and the difference of absolute PSI level between them is detected with Mann-Whitney test. p < 0.05 is considered statistically significant.
(E and F) IGV tracks displaying the reads coverage of SP4 (E) and ZNF638 (F) genes in RNA-seq data of control (green), METTL3- (purple), YTHDC1- (golden), SRSF3- (red), and SRSF10 (blue)-deficient HeLa cells as well as the PAR-CLIP-seq data in the left panel. The red triangle in the bottom represents the GGACH sites within m6A peaks, which are located in the cassette exon. The right panel shows RT-PCR results (top) and statistical analysis of the inclusion level of cassette exons (bottom) upon YTHDC1, SRSF3, SRSF10, or METTL3 depletion relative to control. The inclusion level was quantified and shown as mean ± SEM with indicated p values calculated by Student’s t test. The asterisk indicates the nonspecific bands. p < 0.05 is considered statistically significant.
(G) Distribution of YTHDC1, SRSF3, and SRSF10 binding clusters near splice sites (100 nt upstream and 300 nt downstream from 5′ splice sites, 5′ SS; 300 nt upstream and 100 nt downstream from 3′ splice sites, 3′ SS).
(H) Distribution of YTHDC1, SRSF3, and SRSF10 binding clusters along exon regions adjacent to splice sites (200 nt exon region adjacent to splice sites).
See also Figure S1.
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4. Figure 2 SRSF3 and SRSF10 Competitively Bind to YTHDC1
(A) In vitro pull-down assay using purified Myc-YTHDC1, FLAG-SRSF3, and FLAG-SRSF10 revealed direct interactions between YTHDC1 and these two SR proteins. See also Figures S2A and S2B.
(B and C) In vitro pull-down assay using purified full-length N- or C-terminal truncated proteins revealed that C terminus of both SRSF3 and SRSF10 was involved in their interaction with YTHDC1 (B), and YTHDC1 interacts with both SRSF3 and SRSF10 through its N terminus (C). See also Figures S2A and S2E.
(D and E) Competitive binding of SRSF3 and SRSF10 to YTHDC1. Equal amount of Myc-YTHDC1 protein was immobilized on anti-Myc beads and then incubated with purified equal amount of FLAG-SRSF3 together with an increasing amount of FLAG-SRSF10 (D) or equal amount of FLAG-SRSF10 together with an increasing amount of FLAG-SRSF3 (E). The blots were detected with anti-Myc or anti-FLAG antibody. See also Figures S2G and S2H.
See also Figure S2.
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5. Figure 3 YTHDC1 Regulates Subcellular Localization of SRSF3 and SRSF10
(A and B) Nuclear speckle localization of SRSF3 was decreased (A) while SRSF10 was increased (B) upon siRNA-mediated YTHDC1 depletion, as indicated by immunofluorescence (left). Line scan graphs of ASF (green) and SRSF3/SRSF10 (red) fluorescence intensities in the control or YTHDC1 knockdown cells are shown (right). Scale bar, 10 μm. See also Figures S3A–S3F, S3H, and S3I.
(C and D) Nuclear speckle staining changes of SRSF3 (C) and SRSF10 (D) upon YTHDC1 knockdown can only be restored to the control level by reconstitution with the wild-type, but not m6A-binding-defective, YTHDC1. Arrows indicate GFP-construct-transfected cells. See also Figure S3J.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 YTHDC1 Promotes SRSF3 but Inhibits SRSF10 RNA-Binding Ability
(A–E) Chemiluminescent assay, western blotting (left), and quantitative analyses (right) of total RNAs and m6A-modified RNAs pulled down by FLAG-SRSF3 and FLAG-SRSF10 in the control and YTHDC1-deficient HeLa cells (A); total RNAs and m6A-modified RNAs pulled down by Myc-YTHDC1 or FLAG-SRSF10 in the control or SRSF3-deficient HeLa cells (B); total RNAs and m6A-modified RNAs pulled down by Myc-YTHDC1 or FLAG-SRSF3 in the control or SRSF10-deficient HeLa cells (C); total RNAs pulled down by Myc-YTHDC1, FLAG-SRSF3 or FLAG-SRSF10 in the control and METTL3-deficient HeLa cells (D); and total RNAs pulled down by Myc-YTHDC1 wild-type (Myc-YTHDC1-WT) or double mutant (Myc-YTHDC1-DM, W377A and W428A, which are key m6A-binding amino acids) (E) in HeLa cells. Immunoblot signal intensities of the total RNAs bound by YTHDC1, SRSF3, and SRSF10 were quantified and shown as mean ± SEM with indicated p values calculated by Student’s t test. p < 0.05 is considered statistically significant.
See also Figure S4.
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7. Figure 5
Restored SRSF3/SRSF10 RNA-Binding Ability and AS Patterns in YTHDC1-Depleted Cells by Reconstitution with Wild-Type, but Not m6A-Binding-Defective, YTHDC1
(A and B) Chemiluminescent detection of RNAs pulled down by FLAG-SRSF3 or FLAG-SRSF10 in HeLa cells transfected with (1) siControl and empty Myc expression vector (Myc-EV), (2) siYTHDC1 and Myc-EV, (3) siYTHDC1 and siYTHDC1-insensitive wild-type Myc-YTHDC1 (Myc-YTHDC1-WT-Ins), and (4) siYTHDC1 and siYTHDC1-insensitive double-mutant Myc-YTHDC1 (Myc-YTHDC1-DM-Ins). RNA-binding ability of SRSF3 was decreased significantly in YTHDC1-deficient cells and restored to the control level by overexpressing Myc-YTHDC1-WT-Ins, but not Myc-YTHDC1-DM-Ins (A). RNA-binding ability of SRSF10 was increased significantly in YTHDC1-depleted cells and could be suppressed to the control level by overexpressing Myc-YTHDC1-WT-Ins, but not Myc-YTHDC1-DM-Ins (B). See also Figures S5A and S5B.
(C) The inclusion level of cassette exon in SP4, ZNF638, and ALG11 was validated by RT-PCR in the HeLa cells transfected with (1) siControl and Myc-EV, (2) siYTHDC1 and Myc-EV, (3) siYTHDC1 and Myc-YTHDC1-WT-Ins, and (4) siYTHDC1 and Myc-YTHDC1-DM-Ins. The asterisk indicates the nonspecific bands. See also Figure S5C.
(D) AS patterns of exogenous pSpliceExpress-ZNF638 Exon 2 minigene are consistent with endogenous wild-type ZNF638 gene upon individual knockdown of YTHDC1, SRSF3, or SRSF10. p < 0.05 is considered statistically significant. See also Figures S5D and S5E.
(E) AS patterns of pSpliceExpress-ZNF638 Exon 2 minigene were detected upon expression of the wild-type or YTHDC1/SRSF3/SRSF10 binding-site-mutated (BSM) pSpliceExpress-ZNF638 Exon 2 minigenes. See also Figures S5D and S5F.
Values and error bars in all bar plots represent the mean ± SEM with indicated p values calculated by Student’s t test. p < 0.05 is considered statistically significant.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
YTHDC1 m6A Binding Regulates Exon Inclusion by Recruiting SRSF3; the Effect Could Be Counteracted by SRSF10
(A and B) Western blotting detection and quantification of endogenous-protein level of YTHDC1, SRSF3, and SRSF10. The measurement of endogenous-protein level of YTHDC1, SRSF3, or SRSF10 from WCE (whole-cell extract) according to individual purified protein by western blotting (A). The quantification results (B, upper panel) and relative endogenous-protein level of SRSF3 and SRSF10 compared with YTHDC1 (B, lower panel). The loading of specific amount of purified protein was used as a reference for quantification. Error bars represent mean ± SEM. p < 0.05 is considered statistically significant.
(C) YTHDC1 binds to m6A and by forming a complex with SRSF3, brings SRSF3 to its mRNA-binding regions near m6A, which results in the SRSF3′s binding to exons with m6A modifications and, meanwhile, the probable blockage of SRSF10’s binding to its own mRNA motif leading to the outcome of preferred exon inclusion. In another likely way, SRSF10 may bind to its mRNA region near m6A, which in turn blocks the access of YTHDC1/SRSF3 complex to m6A sites and consequently results in exon skipping. In normal physiological conditions, exon inclusion regulated by YTHDC1 and SRSF3 is predominantly owing to a much higher cellular abundance of SRSF3 protein over SRSF10. Without m6A modification or YTHDC1, SRSF10 may bind to its target mRNAs regions and modulate exon skipping.
Molecular Cell  , DOI: ( /j.molcel )
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