Fig. 2: YTHDC1 recognized the m6A modification of lnc668 to promote the METTL3-mediated m6A modification of lnc668. | Cell Death & Disease

Fig. 2: YTHDC1 recognized the m6A modification of lnc668 to promote the METTL3-mediated m6A modification of lnc668.

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. 2

a MeRIP-qPCR confirmed the presence of m6A modification in lnc668, along with increased methylation after TGF-β1 stimulation. The IgG group served as a negative control to exclude any background signal from nonspecific binding to the antibody. b MeRIP-qPCR confirmed that the methylation level in the over lnc668-WT group significantly increased, whereas that in the over lnc668-Mut 3 groups decreased. The mutation at position 555 led to a significant reduction in m6A methylation in a METTL3-dependent manner. c Western blot analysis confirmed that in the TGF-β1 group, the expression levels of METTL3, METTL14, YTHDC1, and YTHDF2 increased, whereas those of FTO and ALBKH5 decreased. d MeRIP experiments revealed that si-METTL3 and si-METTL14 reduced the m6A modification of lnc668. e qRT-PCR results verified that METTL3, METTL14, and YTHDC1 overexpression increased lnc668 expression, whereas si-METTL3, si-METTL14, and si-YTHDC1 reduced lnc668 expression. f ChIP-PCR results revealed that histone H3K9la was highly enriched in the promoter region of METTL3 in the TGF-β1 group. g qRT-PCR revealed that compared with TGF-β1 stimulation, H3K9la inhibition by si-p300 diminished METTL3 expression. h qRT-PCR results illustrated that lnc668 stability significantly increased in the METTL3 and YTHDC1 overexpression group but significantly decreased in the interference group. i Co-IP experiments enriched the YTHDC1 protein, and Western blot analysis discovered high expression levels of YTHDC1 and METTL3 in the TGF-β1 group. Their interaction was observed in the IP group. j Immunofluorescence exhibited that the colocalization of YTHDC1 and METTL3 increased in the nuclei. k Rescue experiments confirmed that METTL3 overexpression increased lnc668 expression. Interference with YTHDC1 reduced lnc668 expression and reversed the effect of METTL3 on lnc668 expression. l RIP-qPCR experiments indicated that the binding of YTHDC1 with lnc668 increased after TGF-β1 stimulation. The amount of bound lnc668 in the lnc668 overexpression-WT group was greater than that in the TGF-β1 stimulation group. The enrichment of lnc668 significantly reduced in the lnc668 overexpression-Mut group. m RIP experiments showed that METTL3 interference significantly reduced YTHDC1 binding to lnc668, even with lnc668 overexpression.

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