Fig. 7

Roquin induces mRNA decay and inhibits translation of its targets. a Degradation kinetics of reporter mRNAs in HeLa cells transfected with plasmids encoding the CDS of β-globin (βG) without UTR or fused to the Nfkbid 3′-UTR (1–263) without mutation or containing single or combined LMs in the indicated SLs. The amounts of mRNA were quantified by RT-qPCR at the indicated time points after blocking gene-specific transcription with doxycycline. mRNA half-life was calculated with GraphPad Prism. b Representative polysome profiles of βG reporter mRNAs in HeLa cells transfected with plasmids encoding βG without UTR, with the wild-type Nfkbid 3′-UTR (1–263) or Nfkbid 3′-UTR (1–263) containing a single LM in SL2. The amounts of mRNA are shown in percent of the sum detected in all fractions. c The amounts of βG reporter mRNAs in HeLa cells transfected with the same plasmids as in (a) in subpolysomal (fractions 2–5) and polysomal fractions (fractions 6–10). The mRNA amounts were calculated as in Fig. 6c, e. d Scatter plot showing the correlation between mRNA and RPF fold-change (log2) upon overexpression of Roquin-1 in Rc3h1–2^−/− MEF cells (±dox). Cells were treated with doxycycline for 14 h. Red data points represent PAR-CLIP-identified Roquin target mRNAs and black data points show all other cellular mRNAs. e, f Cumulative distributions of mRNA and RPF (e), or TE (f) fold-changes in Roquin-1 overexpressing (+dox) compared to Rc3h1–2^−/− (−dox) MEF cells for two subsets of 500 PAR-CLIP-identified Roquin target mRNAs (red) and nontargets (black). The comparison between target and nontarget distributions was performed with the Mann–Whitney U test. P values for the two-tailed test are indicated. (g, h) mRNA (red) and RPF (blue) fold-change (mean ± SEM) after doxycycline treatment in 100-nt bins spanning the first and last 1000 nt of the ORF of nontargets and all 974 targets (g) as well as 96 translationally-repressed targets of Roquin, defined as the top 10% targets exhibiting greater TE inhibition (h). Data are representative of three (a), two (b, c), and 13 (d–h) independent experiments