The m5C reader Ybx1 regulates embryonic cortical neurogenesis by promoting progenitor cell cycle progression
Fig 5
Ybx1 controls the stability of its m5C-modified target transcripts in neural progenitor cells.
(A-C) Gene Ontology (GO) analysis of transcripts with altered expression levels in E13.5 Ybx1 cKO cortex (A), E18.5 Ybx1 cKO cortex (B), and E13.5 Ybx1 KD cortical progenitors (C). GO terms in biological processes were shown. (D) GO analysis of Ybx1 target transcripts identified by anti-Ybx1 RIP-seq in the E14.5 mouse cortex. (E) Venn diagram showing the overlap of target mRNAs identified by three cKO or KD RNA-seq and anti-Ybx1 RIP-seq. GO terms in biological processes were shown. (F) Validation of m5C modification on Ybx1 target mRNAs by anti-m5C immunoprecipitation combined with RT-qPCR. In vitro transcribed GFP mRNA which has no m5C modification was used as a negative control. Data represent mean ± SD (n = 3 replicates): ***p = 5.99E−04 for Ccnd2; ***p = 1.37E−04 for eEF1g; **p = 0.0045 for Rpsa; ***p = 8.89E−04 for Rps5; ***p = 8.06E−04 for Uhmk1; not significant (ns) for GFP; by unpaired Student t test. (G) Ybx1 target transcripts exhibit accelerated degradation in the Ybx1 cKO cortex. Radial glial cells (RGCs) dissected from E14.5 Ybx1 cKO and control embryos were cultured, treated with actinomycin D (ActD), and collected at different time points. Ybx1 target mRNA levels were measured by RT-qPCR while β-actin mRNA was used a non-target control. Data represent mean ± SEM (n = 3 replicates): for Ccnd2, **p = 0.0017 (2.5 h), ***p = 7.50E−04 (5 h), ***p = 2.32E−04 (7.5 h), **p = 0.0018 (10 h); for eEF1g, **p = 0.0014 (2.5 h), ***p = 3.98E−04 (5 h), ***p = 3.10E−04 (7.5 h), **p = 0.0022 (10 h); for Rpsa, **p = 0.0096 (2.5 h), **p = 0.0012 (5 h), ***p = 4.09E−04 (7.5 h), ***p = 5.08E−04 (10 h); for Rps5, ***p = 5.67E−04 (2.5 h), ***p = 1.33E−04 (5 h), ***p = 1.65E−04 (7.5 h), ***p = 6.41E−04 (10 h); for Uhmk1, **p = 0.0013 (2.5 h), **p = 0.0025 (5 h), **p = 0.0011 (7.5 h), ***p = 7.91E−04 (10 h); for β-actin, ns, not significant; by unpaired Student t test. The half-lives for each mRNA in Ctrl and cKO were calculated (? indicates that the half-lives cannot be calculated). The data underlying all the graphs shown in the figure are included in S1 Data.