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In the screen, each gene was targeted by a pool of four individual si RNAs, and multiplexed flow cytometry was employed during data acquisition (Supplementary Fig. In total, we screened 1495 si RNAs specifically targeting genes encoding proteins associated with four ontology terms: “RNA-binding proteins”, scored positively and thereby validated our screen.Yet, REGNASE-1, an endoribonuclease that cooperates with Roquin-1 in the repression of a reporter containing the most 3′ located 260 nts of the TNF 3′-UTR (termed CDE, was not identified in our screen.It is likely that Roquin induces post-transcriptional repression as part of higher-order messenger ribonucleoprotein particles (m RNPs) that can be regulated in cell-type specific and dynamic ways and differ among the cellular target m RNAs.

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a Immunoblot analysis of Roquin-1, Roquin-2, and α-Tubulin expression or b flow cytometry of ICOS and m Cherry expression in He La reporter cells containing cassettes for stable ICOS and doxycycline-inducible Roquin-1-P2A-m Cherry overexpression.

Cells were either treated with doxycycline (dox) for 18 h or left untreated. d Distribution of ICOS mean fluorescence intensity (MFI) in He La reporter cells after transfection with Roquin-1-targeting si RNA pools (si Rc3h1) or non-targeting control si RNAs (si Ctrl) in a 96-well plate.

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