Figure 7.

m⁵C recognition by ALYREF promotes mRNA export. (A, B) qPCR analysis of the relative cytoplasmic to nucleic ratios of NSUN2 target genes with m⁵C modification in NSUN2 (A) or ALYFEF (B) knockdown HeLa cells reconstituted with control vector, wild-type or mutant forms of NSUN2 (A) or ALYREF (B). Error bars indicate SEM (n = 3). (C-E) FISH analysis of EGFP-tagged FBXW9 Exon 1 minigene mRNAs (red color) in NSUN2 or ALYREF knockdown HeLa cells transfected with m⁵C site-containing wild-type FBXW9-EGFP minigene construct (FBXW9-EGFP-WT, C); line scan graphs (D) and peak density quantification of line scan graphs (E) for minigene mRNAs are shown. The minigene mRNA export was measured by Cy3-labeled oligonucleotide probes complementary to EGFP mRNAs. Scale bar, 10 μm. Error bars indicate SEM (n = 110). (F) Nuclear export of FBXW9 minigene mRNAs in HeLa cells transfected with m⁵C site-containing (FBXW9-EGFP-WT) or mutant (FBXW9-EGFP-MUT) minigene plasmids was analyzed by FISH assay using Cy3-labeled oligonucleotide probes complementary to EGFP mRNAs. (G-H) Line scan graphs (G) and peak density quantification of line scan graphs for minigene mRNAs (H) are shown. Scale bar, 10 μm. Error bars indicate SEM (n = 110). P values were calculated by Student's t-test.