Figure 2. YTHDC1 mediates subcellular localization of methylated mRNAs.

(A) Quantification of m⁶A methylation in total (T), nuclear (N), and cytoplasmic (C) mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (B) Representative western blot of YTHDF2 and members of the mRNA export pathway following knockdown of YTHDC1. (C) Representative analysis of polyA imaging. Nuclei were defined using DAPI signal. Cytoplasmic regions were defined by subtracting nuclear signal from total signal, as defined by DIC imaging. (D) Quantification of polyA signal density. n = 25, two-sided t-test. (E,F) Quantification of m⁶A methylation in nuclear and cytoplasmic mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.

Figure 2—figure supplement 1. YTHDC1 does not contribute to nuclear mRNA decay or transcription.

(A) Representative western blot for YTHDC1 overexpression. (B) Representative western blot for YTHDC1 overexpression in a YTHDF2 knockdown background. (C) Interactions between YTHDC1 and components of the NEXT/PAXT machinery. (D) m⁶A/A ratios in pre-mRNA following knockdown of YTHDC1 and NEXT/PAXT components. (E) Representative western blots of knockdown samples for panel (D). (F) Representative immunofluorescence of YTHDC1–FLAG in HeLa cells. (G) Dot-blot of nascent RNA. HeLa cells were labeled for 1 hr with 4-thio-uridine (4SU), conjugated to biotin and exposed with Streptavidin–HRP. (H) Methylene blue staining of (G) (loading control).