Skip to main content
. 2021 May 15;41(7):560–575. doi: 10.1002/cac2.12158

FIGURE 2.

FIGURE 2

ALYREF directly binds to PKM2 mRNA and regulates its stability in an m5C‐dependent manner. (A) The RIP assays show that ALYREF interactwith 3′‐UTR of PKM2 in T24 cells. (B) The RIP assays were performed in T24 cells transfected with ALYREF wild type or K171A mutation. qRT‐PCR assays show that K171A mutation result in a significantly decreased level of ALYREF binding to 3′‐UTR of PKM2. (C) m5C‐modified RNAs were detected by dot blotting in T24 cells with or without NSUN2 knockdown. NSUN2 knockdown significantly decreases the m5C level of total RNAs. MB was used as a loading control. (D) NSUN2 knockdown dramatically decreases the m5C level of the 3′‐UTR of PKM2 in T24 cells by MeRIP assay. (E) RIP assays reveal the binding efficiency of ALYREF to PKM2 3’‐UTR is decreased following knockout of NSUN2 in T24 cells. (F, G) ALYREF overexpression promotes PKM2 expression, while ALYREF with K171A mutation fails to regulate PKM2 expression as qualified by qRT‐PCR and Western blotting. (H, I) ALYREF knockdown decreases PKM2 expression in 5637 and T24 cells as measured by qRT‐PCR and Western blotting. (J) Relative luciferase activity of the reporter carrying PKM2 3′‐UTR with either wild‐type or m5C site deletion co‐transfected with ALYREF (ALY‐WT), ALYREF K171A mutant (ALY‐MUT), or control vector (GFP) into 293T cells was detected and normalized to renilla luciferase activity. (K) ALYREF protein is located in both nucleus (green arrows) and cytoplasm (white arrows) of 5637 and T24 cells by immunofluorescence assays. (L) 5637 and T24 cells were treated with Act D. The half‐life of PKM2 mRNA can be shortened by ALYREF knockdown and lengthened by ALYREF overexpression. ALYREF with K171A mutation can not regulate the half‐life of PKM2 mRNA. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ALYREF, Aly/REF export factor; PKM2, Pyruvate kinase muscle isozyme M2; m5C, 5‐methylcytidine; RIP, RNA immunoprecipitation; UTR, untranslated region; qRT‐PCR, quantitative real‐time polymerase chain reaction; NC, negative control; shNC, negative control of knockdown; NSUN2, NOP2/Sun RNA methyltransferase family member 2; shNSUN2, knockdown of NOP2/Sun RNA methyltransferase family member 2; shALYREF, knockdown of Aly/REF export factor; FPKM, fragments per kilobase million values; MB, methylene blue; SV40, simian virus 40; hLuc, fireflyer luciferase; PA, poly A; CMV, cytomegalovirus; Rluc, Renillar luciferase; PKM2‐WT, Pyruvate kinase muscle isozyme M2 wild type; PKM2‐WUT, Pyruvate kinase muscle isozyme M2 mutation; Act D, actinomycin D.