Fig. 4

MiR-b2122 reduces firefly luciferase activity when it contains either the TARDBP, FUS/TLS or RGNEF 3’UTR. HEK293T cells were transfected with a pmirGLO plasmid containing the 3’UTR of one of the RNA-binding proteins of interest either with or without miR-194 or miR-b2122. PmirGLO plasmid without any 3’UTRs were also transfected with or without miRNAs of interest to determine the miRNAs effect on the plasmid itself. a Reporter gene assay revealed miR-b2122 reduced firefly activity when it contained either the TARDBP, FUS/TLS, or RGNEF-short/long 3’UTR, whereas miR-194 down-regulated firefly levels when it contained either TARDBP, FUS/TLS, but not RGNEF-long. MiR-194 has no MRE in RGNEF-short, and thus, the interaction between the two was not examined. b RT-qPCR results showed similar suppression of mRNA levels as seen to the luciferase activity observed in the reporter gene assay. Let-7a was used as a negative control for these experiments. Firefly was normalized to renilla luciferase, and then further normalized to account for the effect of each miRNA on the pmirGLO vector to determine the exact effect that each miRNA has on each 3’UTR. Each miRNA was compared to its own individual control based on the normalization of the data. Data is expressed as sample mean ± SEM, and significance was determined using a Student’s t-test (*** = p < 0.001, ** = p < 0.01, * = p < 0.05, NS = p > 0.05)