Abstract
The RNA-binding proteins Roquin-1 and Roquin-2 redundantly control gene expression and cell-fate decisions. Here, we show that Roquin not only interacts with stem–loop structures, but also with a linear sequence element present in about half of its targets. Comprehensive analysis of a minimal response element of the Nfkbid 3′-UTR shows that six stem–loop structures cooperate to exert robust and profound post-transcriptional regulation. Only binding of multiple Roquin proteins to several stem–loops exerts full repression, which redundantly involved deadenylation and decapping, but also translational inhibition. Globally, most Roquin targets are regulated by mRNA decay, whereas a small subset, including the Nfat5 mRNA, with more binding sites in their 3′-UTRs, are also subject to translational inhibition. These findings provide insights into how the robustness and magnitude of Roquin-mediated regulation is encoded in complex cis-elements.
Highlights
Background modelThree sets of randomized sequences served as control for the background frequency of various sequence and RNA structural elements.Ribosome profiling and RNA input for sequencing
With the previously described method[22], we found 1121 mRNAs containing sites that were enriched in CLIP reads relative to what is expected from the mRNA expression level across three biological replicates (Supplementary Fig. 1b), 974 of which had a total of 1423 reproducibly-bound sites (Supplementary Data 1 and 2)
Many of these mRNAs overlapped with mRNAs identified by PAR-CLIP in HEK293 cells[14], whereas a smaller fraction of the mRNAs that were co-immunoprecipitated with Roquin-1 from extracts of either IL-1β-stimulated HeLa or LPS-stimulated mouse macrophages were present in our target set (Supplementary Fig. 1c, d)[4,19]
Summary
Background modelThree sets of randomized sequences (obtained by shuffling the PAR-CLIP-derived binding sites) served as control for the background frequency of various sequence and RNA structural elements.Ribosome profiling and RNA input for sequencing. 6. In brief, ribosome profiling was carried out with the ARTseq Ribosome Profiling Kit for mammalian cells, according to the manufacturer’s instructions (Illumina; cat #RPHMR12126). After inactivation of the nuclease with 10 μl of SUPERaseInTM RNase Inhibitor (Invitrogen; cat# AM2694), the 80S monosomes together with other large proteins or protein complexes were purified by size-exclusion chromatography using MicroSpin S-400h columns (GE Healthcare; cat # 27-5140-01), according to the manufacturer’s instructions. CDNA libraries of the RPFs were prepared according to the ARTseq Ribosome Profiling Kit and DNA fragments of the correct size (nt 113 + 28–30) were gel-purified using a nondenaturing 8% polyacyrlamide TBE gel. Total RNA for Ribosome profiling analysis was purified from 200 μl of clarified MEF cell lysate without ARTseq Nuclease treatment using acidic phenol–chloroform, followed by ethanol precipitation. CDNA libraries of RPFs and total RNA were sequenced using an Illumina HighSeq2000 sequencer Total RNA for Ribosome profiling analysis was purified from 200 μl of clarified MEF cell lysate without ARTseq Nuclease treatment using acidic phenol–chloroform, followed by ethanol precipitation. cDNA libraries were performed with 100 ng of total RNA using the Encore Complete RNA-Seq DR Multiplex Systems (NuGEN Technologies cat #0333-32 and cat #0334-32). cDNA libraries of RPFs and total RNA were sequenced using an Illumina HighSeq2000 sequencer
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