Fig. 6: m6A methylation modulates the regulatory action of IMP1 on the microtubule-related targets.

a Gene reporter assay constructs used in the study. Constructs were derived from the PsiCheck-2 vector and include the Renilla and Firefly luciferases ORFs and various 3ʹUTR sequences with m6A methylated IMP1 binding sites. Image created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Mapping of IMP1 iCLIP crosslink signal expressed in counts per millions (top) and m6A sites (dash, bottom) onto the 3ʹUTR of selected genes in neurons. Merged signal from all replicates is shown for iCLIP. The grey squares highlight the sections of the 3ʹUTRs used for the Luciferase assay experiments, which contain IMP1 binding sites overlapping with m6A sites. m6A sites located in the cloned regions are highlighted in red. c Relative luciferase activity was determined by a dual-luciferase assay system. Rluc-MAP2-UTR3 or Rluc-DCX-UTR3 constructs were co-transfected in HeLa cells with (1) siRNA CTRL, (2) siRNA CTRL + sRNA IMP1, (3) siRNA CTRL + siRNA METTL3, (4) siRNA IMP1 and siRNA METTL3, (5) siRNA CTRL with IMP1 expressing vector, or (6) siRNA METTL3 with IMP1 expressing vector. For MAP2 n = 8 replicates (4 independent experiments), for DCX n = 8 replicates for conditions 1, 2 and 3 (4 independent experiments), n = 6 and 7, respectively for conditions 5 and 6 (3–4 independent experiments). Data is presented as the mean ± SEM. Where data were found to be normally distributed, a two-sided unpaired Student’s t test was employed; where at least one of the datasets were not normally distributed when comparing 2 conditions, a two-sided Mann–Whitney U test was used. P values are displayed on the plot. RL stands for Renilla luciferase and FL is for firefly luciferase. d Western blot analysis showing expression of IMP1, METTL3, TUBB4A, MAP2, DCX and H3 as loading control in neurons treated with non-targeting control siRNA (siCTRL) or siRNA targeting METTL3 (siMETTL3). Representative images from 2 independent experiments performed on three technical replicates from 2 independent iPSC lines (biological replicates). e A working model for m6A regulation of IMP1 binding during development. As IMP1 abundance during differentiation decreases, increased m6A methylation of a subset of targets favours the selective recruitment of the protein. Image created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.