Figure 5. YTHDC1 mediates export of m6A-modified mRNA through SRSF3 and NXF1.

(A) Immunoprecipitation (IP) of endogenous YTHDC1 from HeLa cell lysate. (B) IP of endogenous SRSF3 and NXF1 from HeLa cell lysate. (C) LC-MS/MS of mRNA cross-linked to SRSF3 and NXF1. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (D) Quantification of m⁶A in nuclear mRNA following knockdown of SR proteins. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.

Figure 5—figure supplement 1. YTHDC1 interacts with export-competent SRSF3.

(A) Immunoprecipitation (IP) of YTHDC1-FLAG with members of the SR protein family. (B) IP of YTHDC1 C- and N-terminal regions with phosphorylated SRSF3. (C) Coomassie stain of His-SRSF3-RRM purified from Escherichia coli. (D) In vitro IP of HeLa mRNA with His-SRSF3-RRM. Error bars represent mean ± standard deviation, n = 4 (two biological x two technical replicates), two-sided t-test of equal variance.

Figure 5—figure supplement 2. Roles of SRSF3 and other SR proteins in the export of m⁶A-methylated mRNAs.

(A,B) Full data from Figure 5D. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two sided t-test with equal variance. (C) Representative western blots for siRNA knockdown performed for Figure 5D. (D) PolyA imaging of HeLa cells following treatment with siControl, siYTHDC1, or siSRSF3. Yellow outlines the nuclear regions (defined by DAPI signal) and the cytoplasmic regions (defined by DIC). (E) m⁶A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. Curves fit to exponential decay.

Figure 5—figure supplement 3. Estimate of relative protein stoichiometry.

(A) Fragments per kilobase of transcript per million (FPKM) from total RNA-seq (average of two biological replicates). (B) Estimate of protein copies per cell in HeLa cells (Nagaraj et al., 2011).

Figure 5—figure supplement 4. Subcellular distribution of YTHDC1 target mRNAs as a function of YTHDC1, SRSF3, and m⁶A.

(A) Representative western blot for simultaneous siRNA knockdown of METTL3 and METTL14 in HeLa cells. (B) RT-PCR of nuclear RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. (C) RT-PCR of cytoplasmic RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance.