Fig. 6: YBX1 and YBX3 preferentially recognize and stabilize mRNAs with NSUN6-mediated m5C modifications.
From: A cohort of mRNAs undergo high-stoichiometry NSUN6-mediated site-specific m5C modification
a Schematic diagram showing the flowchart of the RNA pull-down experiment. Biotin-labeled FURIN-25nt and FURIN-25nt-m5C were incubated with the cytoplasmic lysate of MDA-MB-231 cells. Proteins enriched by streptavidin were analyzed using mass spectrometry. b Scatter plot showing proteins binding to FURIN-25nt-m5C versus FURIN-25nt RNA in MDA-MB-231 cells. Potential reader proteins YBX1 and YBX3 were highlighted in red. Western blot analysis of the FLAG-YBX1 (c) or FLAG-YBX3 (d) pulled down by biotin-labeled FURIN-25nt or FURIN-25nt-m5C in HEK293T cells (left panels) and the corresponding quantification (right panels).Biolayer interferometry analysis of RNA binding affinity of the YBX1 CSD (e), and YBX3 CSD (f) with unmodified FURIN-25nt and FURIN-25nt-m5C. g The structural model of the YBX3 CSD and PVRL2-25nt complex generated using AlphaFold3. PVRL2-25nt was in dark blue, and the YBX3 CSD was in yellow. h Recognition of m5C by YBX3 in the AlphaFold3-generated YBX3 CSD and PVRL2-25nt complex. Hydrogen bonds were indicated with white dashes, and residues involved in the recognition of m5C were labeled. i The Venn diagram of the overlap between mRNA substrates of NSUN6 and mRNAs targeted by YBX1 and YBX3 from the published CLIP datasets31,62. The RT-qRCR analysis of mRNA half-lives following YBX1 (j) or YBX3 (k) knockdown in MDA-MB-231 cells. l The RT-qRCR analysis of mRNA half-lives in YBX1 knockdown MDA-MB-231 cells overexpressing either YBX1 WT or the YBX1 W65F mutant. m The RT-qRCR analysis of mRNA half-lives in YBX3 knockdown MDA-MB-231 cells overexpressing either YBX3 WT or the YBX3 W97F mutant. Data in c–f, and j–m are presented as the mean ± SD for three independent experiments. p-values are from two-tailed t test (c, d) or one-tailed t test (e, f). ***p < 0.001, **p < 0.01, *p < 0.05. n A simplified model shows the biogenesis and function of NSUN6-mediated high-stoichiometry m5C modifications on a cohort of cell migration-related mRNAs in breast cancer cells. Created in BioRender. Zhang, Y. (2025) https://BioRender.com/bk4f7pg. Source data are provided as a Source Data File.
