Fig. 6: Functional rare 3′ UTR variants regulate mRNA stability and cell proliferation in HEK293T cells. | Nature Communications

Fig. 6: Functional rare 3′ UTR variants regulate mRNA stability and cell proliferation in HEK293T cells.

From: Massively parallel screen uncovers many rare 3′ UTR variants regulating mRNA abundance of cancer driver genes

Fig. 6

a MapUTR activity scores of functional variants (MFN2: rs777822288, FOSL2: rs11884725, and IRAK1: rs782486025) measured in HEK293 cells. P-values were calculated using MPRAnalyze’s two-sided likelihood ratio test and corrected using the Benjamini–Hochberg method (N = 3 biologically independent samples). *p < 0.05, **p < 0.01, ***p < 0.001. b Experimental validation workflow. HEK293T cells were transfected with plasmids expressing PEmax and epegRNAs to introduce the variant of interest. Single-cell clones were isolated and genotyped as either reference (Ref) or variant (Var) clones. The single clones were then used for downstream assays to test for mRNA expression/stability or cell proliferation. Created with BioRender.com. c–e Sanger sequencing results confirming the genotype of single clones with reference (Ref) and variant (Var) alleles for MFN2 (c), FOSL2 (d), and IRAK1 (e). The alleles are underlined. f–h Normalized mRNA expression level of reference (Ref) and variant (Var) alleles of MFN2 (f), FOSL2 (g) and IRAK1 (h). Cells were treated with 10 μg/ml actinomycin D (ActD) and harvested at 2 h, 8 h, and 24 h post-treatment to test for mRNA stability. For MFN2, six biological replicates (N = 6 clones) for each allele were included in the experiment. For FOSL2 and IRAK1, four biological replicates (N = 4 clones) per allele were included. P-values were calculated using one-tailed Student’s t test. *p < 0.05. i–k Cell proliferation assay of single clones with reference (Ref) and variant (Var) alleles for MFN2 (i), FOSL2 (j), and IRAK1 (k). For MFN2, six biological replicates (N = 6 clones) for each allele were included in the experiment. For FOSL2 and IRAK1, four biological replicates (N = 4 clones) per allele were included. The vertical dashed line indicates the cell confluence values at 48 h, which are plotted on the right. P-values were calculated using one-tailed Student’s t test. *p < 0.05, **p < 0.01. a, f–k Boxplots are plotted as median, the 25% and 75% percentiles, and non-outlier maxima and minima. Specific p-values are provided in the Source Data file.

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