Fig. 5
From: Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq
Sfpq promotes post-transcriptional silencing mediated by miRNAs in both nucleoplasm and cytoplasm. a Gene expression differences in let-7a-transfected P19 cells upon Sfpq knockdown and control. Differential expression is plotted for mRNAs containing canonical let-7a-binding sites (BS) localized in the 3′UTR within a close distance to Sfpq peaks (<500 nt) and reduced upon Sfpq knockdown. b P19 cells were transfected with the indicated molecules. Nucleoplasm and cytoplasm were separated to measure the expression levels of the indicated let-7a-target mRNAs. RNA was purified and analyzed by RT-qPCR. Data are normalized with U2 snRNA and presented as the mean ± s.e.m. (n = 3). c Immunoblot analysis of Lin28A and tubulin in P19 cells transfected with let-7a and/or siSfpq. d Quantitative RT-PCR analysis of the half-life of Lin28A transcript in let-7a-transfected P19 cells compared to control (upper panels) or siSfpq + let-7a-transfected cells compared to siSfpq control (lower panels). Total RNA from either nucleoplasm (left panels) or cytoplasm (right panels) was isolated at the indicated times after addition of actinomycin D. Data are normalized with β2-microglobulin and presented as the mean ± s.e.m. (n = 6). e Relative luciferase activity of reporter constructs containing the mouse Lin28A 3′UTR sequence in HEK293T cells transfected with let-7a and siSfpq as indicated. The data were normalized using Renilla activity and presented as the mean ± s.e.m. (n = 4). One-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, ns not significant
