Fig 2: Involvement of m5C in mRNA export regulation. (A-C) Fluorescence in situ hybridization (FISH) analysis of mRNAs (red) in the control, NSUN2- or ALYREF-de?cient HeLa cells (A); line scan graphs (B) and peak density quantification of line scan graphs (C) for mRNAs are shown. Green: FAM-labeled siRNAs. The red and black dash lines (B) represent the peak densities of nuclear and cytoplasmic mRNAs, respectively. Scale bar, 10 µm. Error bars indicate SEM (n = 120). (D-I) FISH analysis of mRNAs (red color) in NSUN2 (D-F) or ALYREF (G-I) knockdown HeLa cells reconstituted with control vector, EGFP/GFP-tagged wild-type or mutant forms of NSUN2 (D-F) or ALYREF (G-I); line scan graphs (E, H) and peak density quantification of line scan graphs (F, I) for mRNAs are shown. The red and black dash lines (E, H) represent the peak densities of nuclear and cytoplasmic mRNAs, respectively. Scale bar, 10 µm. Error bars indicate SEM (n = 120). P values were calculated by Student's t-test.

Fig 3: ALYREF is a specifi c mRNA m5C-binding protein. (A) Scatter plot of proteins bound to Oligo-m5C versus Oligo-C RNA oligos. The plot was based on the average peptide numbers of proteins detected in both replicates. Enriched ALYREF protein was highlighted. (B) Demonstration of endogenous ALYREF pulled down by biotin-labeled RNA oligonucleotides containing m5C (Oligo-m5C). Left, western blotting; right, quantification level. (C) Demonstration of purified Flag-ALYREF pulled down by biotin-labeled Oligo-m5C. Left, western blotting; right, quantification level. (D) EMSA (left) and line graph quantification (right) showing the binding ability of purified Flag-ALYREF-WT with Oligo-m5C or Oligo-C. 100 nM of RNA Oligo-m5C or Oligo-C was incubated with different concentrations of Flag-ALYREF-WT protein. The RNA binding ratio was calculated by (RNA-protein)/((free RNA) + (RNA-protein)). Error bars indicate SEM (n = 3). P values were calculated using Student's t-test. (E) UHPLC-MRM-MS/MS chromatograms (left) and quantification (right) of m5C in input and in vitro ALYREF-RIP mRNA samples. (F) Boxplot showing m5C level of methylation sites detected in both input and in vivo ALYREF-RIP mRNA samples. P values were calculated using Mann-Whitney U test.

Fig 4: MTR4 regulates the release of MAT2A mRNA from the nucleus. (A) Schematic diagram of the fate of RNA in the nucleus. If an mRNA can effectively recruit ALYREF, it will exit the nucleus smoothly and efficiently. Conversely, MTR4 may bind to ARS2, and then recruit exosomes to degrade it. (B) Western blotting for MAT2A, MTR4, and ALYREF in U251-M and U251 cells following culture in CM or MRM. ß-actin was used as an internal control. (C) U251 cells were transfected with MTR4, and immunoblotting analysis was performed with the indicated antibodies. (D,E) U251 and U87 cells were cultured with MRM for 0, 4, 8, 12, and 24 h; the cytoplasm and nucleus were separated; and cytoplasm mRNA levels of MAT2A were analyzed by qRT-PCR. (F) U251 cells were transfected with MTR4 (0, 1, 2, 3, or 4 µg); again the cytoplasm and nucleus were separated, and cytoplasm mRNA levels of MAT2A were analyzed by qRT-PCR. *p < 0.05.

Fig 5: ECD interacts with DDX39A and other components of the mRNA export machinery. (A to E) HEK-293T (A to D) or MCF10A (E) cell lysates were immunoprecipitated (IP) with the antibodies indicated at the top, followed by Western blotting (WB) with the antibodies shown on the left. ALY and CRM1 were used as positive controls. CBL, mouse IgG (mIgG), and rabbit IgG (rIgG) were used as negative controls; 100-µg aliquots of lysate protein were used in the input lane. (F) Twenty micrograms of GST (negative control) or GST fusion with full-length ECD (1 to 644 aa) was incubated with 1 mg of protein lysate from HEK-293T cells transfected with FLAG-tagged RUVBL1 or FLAG-tagged DDX39A, and the GST pulldown proteins were analyzed by Western blotting with the indicated antibodies. The membrane was stained with Ponceau S to visualize the GST fusion proteins to assess comparable fusion protein use for pulldowns (indicated by arrows). (G) GST or GST fusion with full-length ECD (1 to 644) or indicated ECD mutants were incubated with protein lysate of HEK-293T cells transiently transfected with FLAG-tagged-DDX39A and FLAG-tagged RUVBL1, and the bound proteins were analyzed by Western blotting with the indicated antibodies. The membrane was stained with Ponceau S to visualize the GST fusion proteins (indicated by arrows). The experiment shown is a representative of at least three repeats with comparable results. (H) To validate ECD commercial antibody, Western blotting was performed in various cell lysates from control and ECD siRNA-treated MCF10A, 76NTERT, MDA-MB-231 and HeLa cells and ECD-overexpressing MCF10A stable as well as DOX-inducible (as indicated); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (I) ECD colocalizes with DDX39A in the nucleus. MCF10A cells untreated or treated with 20 ng/µl of leptomycin B (LMB) for 4 h and fixed in 4% paraformaldehyde (PFA) were subjected to immunofluorescence staining using anti-ECD rabbit polyclonal and anti-DDX39 mouse monoclonal antibodies followed by imaging using 63× Zeiss confocal microscope (scale bars, 10 µm).