Fig. 4: MTR4 binds to a large panel of pre-mRNA and regulates the alternative splicing of GLUT1 and PKM genes.

a MTR4 binding motifs, UAAAAAA(U)AA(C)A(U)AA (which is the conventional MTR4 binding region, poly(A) sequences) and CCAG(C/U/G)C, are defined by analyzing sequence of 3301 peaks from RIP-seq with motif analysis tool, MEME-Chip. b, d A schematic of PKM gene (b) or GLUT1 gene (d) displaying potential binding motifs for MTR4 and two RNA alternative splicing factors PTBP1 and hnRNPA. Numbers indicate the locations of primers for PCR. c, e RIP analysis of the binding of MTR4 to the predicted MTR4 binding motifs of PKM gene (c) or GLUT1 gene. e Due to the lack of anti-MTR4 antibodies that can be used for immunoprecipitation, PLC/PRF5 cells expressing HA-tagged MTR4 were subjected to immunoprecipitated with anti-HA antibody or IgG. Eluted RNA was analyzed by RT-qPCR and display as % of input. n = 2 independent experiments. Data are presented as mean values. Numbers indicate the locations of primers for PCR (f, g) Relative mRNA levels of PKM2/PKM1 (f) or GLUT1b/GLUT1a (g) in MTR4 KD cells versus control cells. Dotted line indicates 1. Data are presented as mean values. n = 2 independent experiments. h, i Alternative splicing (AS) of minigenes (mini.) of PKM (h) or GLUT1 (i) genomic DNA containing exons undergoing AS in MTR4 KD or control cells. Primers for PCR analysis are indicated by arrows. Data are presented as mean value ± s.d. Two-tailed, unpaired t-test. p value is indicated. n = 3 independent experiments. j, k RIP analysis of MTR4 binding to the WT and MTR4 binding motif mutant PKM minigene (j) or GLUT1 minigene (k). PLC/PRF5 cells expressing HA-tagged MTR4 in combination with vector expressing indicated minigenes were subjected to immunoprecipitation with anti-HA antibody or IgG. Eluted RNA was analyzed by RT-qPCR and display as fold changes over IgG. Data are presented as mean values. n = 2 independent experiments. Source data are provided as a Source Data file.