Figure 5.

YTHDC1 regulates expressions of MCM genes by stabilizing their mRNAs. (A) Volcano plot showing fold changes for differentially expressed genes in MOLM-13 cells expressed Scramble shRNA, YTHDC1 shRNA1#, and YTHDC1 shRNA2#. The cells were collected 2 to 3 days after viral infection when different cell groups had a similar viability. The experiments were performed in duplicate. (B-C) The bar graph showing the pathways significantly affected in YTHDC1 KD MOLM-13 cells. Downregulated pathways are shown in panel B, and upregulated pathways are shown in panel C. RNA-sequencing analysis was performed by using Partek Flow software. (D) Analysis of RNA-sequencing data, m⁶A-sequencing data, and YTHDC1 RIP-sequencing data to define YTHDC1 direct target genes. (E) qRT-PCR validation of the selected target genes in MOLM-13 cells expressed Scramble shRNA (Scr), YTHDC1 shRNA1# (Ysh1#), and YTHDC1 shRNA2# (Ysh2#). (F) mRNA m⁶A methylation analyses of selected target genes (MCM2, MCM4, MCM5, CHAF1A, and BCL2) by MeRIP assay in MOLM-13 cells. (G) YTHDC1-RIP analyses showing YTHDC1 enrichments at mRNAs of the selected target genes in MOLM-13 cells. (H) mRNA m⁶A methylation analyses of selected target genes (MCM2, MCM4, MCM5, CHAF1A, and BCL2) by MeRIP assay in MOLM-13 cells expressed Scramble shRNA (Scr), YTHDC1 shRNA1# (Ysh1#), and YTHDC1 shRNA2# (Ysh2#). (I) YTHDC1-RIP analysis of YTHDC1 enrichments at mRNAs of the selected target genes in MOLM-13 cells expressed Scramble shRNA (Scr), YTHDC1 shRNA1# (Ysh1#), and YTHDC1 shRNA2# (Ysh2#). mRNA half-life of the selected target genes (MCM2 [J] and MCM4 [K]) in MOLM-13 cells with or without YTHDC1 KD. The P value was detected by 1-way analysis of variance followed by multiple comparisons vs the Scr group 16 hours after actinomycin D treatment. Data are presented as mean ± standard deviation; Student t test. Experiments were performed in triplicate and were repeated at least twice. *P < .05; **P < .01; ***P < .001.