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. 2021 Feb 10;11:3476. doi: 10.1038/s41598-021-82706-2

Figure 3.

Figure 3

MEF2C regulates 3′-uridylation of miRNAs in human muscle cells. (a) The pie charts show the frequency of four types of modifications of the miRNA sequence in differentiated HSkM cells. The experiment was performed in HSkM cells in triplicates. (b) Comparison of the frequency of 5′- and 3′-modifications of miRNAs in control and MEF2C KD cells based on the results of small RNA-seq experiments ( *P < 0.05; **P < 0.01). (c) Comparison of the number and length of 3′-nontemplated U- and A-tailed reads of miRNAs in control and siMEF2C_1 or siMEF2C_2 treated myocytes. The X-axis represents the length of U- or A-tails, and the Y-axis represents the percentile of modified reads in small RNA-seq ( *P < 0.05). (d) Modified polyA-RT-qPCR assays were used to quantify canonical forms of miR-221 and miR-92a (lower panels) and uridylated isoforms of miR-221-U and miR-92a-U (upper panels), in muscle cells with MEF2C deficiency (*P < 0.05, **P < 0.01, ***P < 0.0001). The experiment was performed in triplicates. (e) In vitro uridylation assay of internally radiolabeled pre-miR-27b incubated with extracts from siCTRL- or siMEF2C_1-treated HSkM cells. The mixtures were either supplemented or not supplemented with 0.25 mM UTP (+ or − rUTP). Reaction products were separated by denaturing PAGE, analyzed by autoradiography, and the amount of pre-miRNA short fragments (13–17 nt) was calculated and is shown on a graph (below) as a fraction of unprocessed pre-miRNA (***P < 0.0001). Fully uncut gel images are shown in Supplementary information, uncut images section. (f) Accumulation of short fragments of miRNAs in differentiated HSkM cells upon siMEF2C_1 KD calculated based on RNA-seq data (n = 3 for siCTRL and n = 3 for siMEF2C_1). Alterations in the level of miRNA fragments are shown as fold changes; fold change is defined as the ratio between the counts of short fragments of miRNA in siMEF2C_1 KD and the counts of short fragments of miRNA in siCTRL cells divided by the counts of short fragments of miRNA in siCTRL cells (no change is equal to 1).