Fig. 5: Phase-separating YTHDC1 promoted the nuclear export of lnc668 by forming a transport complex with SRSF3–ALYREF–XPO5. | Cell Death & Disease

Fig. 5: Phase-separating YTHDC1 promoted the nuclear export of lnc668 by forming a transport complex with SRSF3–ALYREF–XPO5.

From: YTHDC1 phase separation drives the nuclear export of m6A-modified lncNONMMUT062668.2 through the transport complex SRSF3–ALYREF–XPO5 to aggravate pulmonary fibrosis

Fig. 5

a YTHDC1 was enriched in Co-IP experiments. Western blot analysis of the whole cell lysate in the Input group revealed low expression levels of YTHDC1 and SRSF3 in the normal group and high expression levels of YTHDC1 and SRSF3 after TGF-β1 stimulation. Western blot analysis of the lysate enriched with YTHDC1 showed low expression levels of YTHDC1 and SRSF3 in the normal group in the IP group and high expression levels of YTHDC1 and SRSF3 after TGF-β1 stimulation. IP: Immunoprecipitation group; IgG: Negative control group; Input: Positive control group. b Immunofluorescence detection revealed that YTHDC1 and SRSF3 colocalized in the nucleus and their colocalization in the TGF-β1 stimulation group increased. c RNA-FISH experiments showed that after the overexpression of YTHDC1, the expression of lnc668 increased in the nucleus and cytoplasm, with a more pronounced increase in the cytoplasm than in the nucleus. si-SRSF3 led to a decrease in the expression of lnc668 in the cytoplasm, reversing the promoting effect of YTHDC1 overexpression. d Immunofluorescence experiments showed that overexpression of YTHDC1-WT increased YTHDC1 and SRSF3 levels, while YTHDC1-Δ274-294 overexpression decreased both proteins. e Co-IP experiments found that the binding of SRSF3 increased after the overexpression of YTHDC1 but decreased after the overexpression of YTHDC1-Δ274-294. f The nuclear-cytoplasmic separation experiments confirmed that TGF-β1 stimulation, the nuclear expression of XPO5 and SRSF3 increased, while interference with YTHDC1 led to a decrease in their nuclear expression. Overexpression of YTHDC1 promoted the nuclear import of XPO5 and SRSF3, whereas overexpression of YTHDC1-Δ274-294 lost the ability to promote the nuclear import of XPO5 and SRSF3. g The nuclear-cytoplasmic separation experiments confirmed that TGF-β1 stimulation increased the nuclear import of ALYREF and its colocalization with SRSF3, while interference with YTHDC1 reduced this colocalization, causing ALYREF to accumulate in the cytoplasm. Overexpression of YTHDC1 enhanced the nuclear colocalization of ALYREF and SRSF3, whereas overexpression of YTHDC1-Δ274-294 resulted in the loss of the ability to promote the nuclear import of ALYREF, leading to increased localization of ALYREF in the cytoplasm. h Immunofluorescence demonstrated that SRSF3 and XPO5 were expressed in the nuclei. After TGF-β1 stimulation, the nuclear expression levels of SRSF3 and XPO5 increased. Interfering with YTHDC1 reduced the nuclear expression of SRSF3 and XPO5, whereas overexpressing YTHDC1 increased the nuclear expression of SRSF3 and XPO5. i Immunofluorescence demonstrated that ALYREF was mainly located in the cytoplasm of normal cells, whereas SRSF3 localized in the nucleus. After stimulation with TGF-β1, the nuclear expression of ALYREF increased and the cytoplasmic expression of ALYREF decreased. The nuclear expression of SRSF3 also increased. Interference with YTHDC1 led to a reduction in the colocalization of ALYREF and SRSF3 in the nucleus. By contrast, the overexpressed YTHDC1 enhanced the nuclear colocalization of these two proteins, whereas the overexpressed YTHDC1-Δ274-294 resulted in the decreased nuclear colocalization of ALYREF and SRSF3 proteins.

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