A Distinct, Sequence-Induced Conformation Is Required for Recognition of the Constitutive Decay Element RNA by Roquin.

The constitutive decay element (CDE) of tumor necrosis factor α (TNF-α) mRNA (Tnf) represents the prototype of a class of RNA motifs that mediate rapid degradation of mRNAs encoding regulators of the immune response and development. CDE-type RNAs are hairpin structures featuring a tri-nucleotide loop. The protein Roquin recognizes CDE-type stem loops and recruits the Ccr4-Caf1-Not deadenylase complex to the mRNA, thereby inducing its decay. Stem recognition does not involve nucleotide bases; however, there is a strong stem sequence requirement for functional CDEs. Here, we present the solution structures of the natural Tnf CDE and of a CDE mutant with impaired Roquin binding. We find that the two CDEs adopt unique and distinct structures in both the loop and the stem, which explains the ability of Roquin to recognize stem loops in a sequence-specific manner. Our findings result in a relaxed consensus motif for prediction of new CDE stem loops.

An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins.

Determining the composition of messenger ribonucleoprotein (mRNP) particles is essential for a comprehensive understanding of the complex mechanisms underlying mRNA regulation, but is technically challenging. Here we present an RNA-based method to identify RNP components using a modified streptavidin (SA)-binding RNA aptamer termed S1m. By optimizing the RNA aptamer S1 in structure and repeat conformation, we improved its affinity for SA and found a 4-fold repeat of S1m (4×S1m) to be more efficient than the established MS2 and PP7 systems from bacteriophages. We then attached the AU-rich element (ARE) of tumor necrosis factor alpha (TNFα), a well-known RNA motif that induces mRNA degradation, via 4×S1m to a SA matrix, and used the resulting RNA affinity column to purify ARE-binding proteins (BPs) from cellular extracts. By quantitative mass spectrometry using differential dimethyl labeling, we identified the majority of established ARE-BPs and detected several RNA-BPs that had previously not been associated with AREs. For two of these proteins, Rbms1 and Roxan, we confirmed specific binding to the TNFα ARE. The optimized 4×S1m aptamer, therefore, provides a powerful tool for the discovery of mRNP components in a single affinity purification step.

Roquin promotes constitutive mRNA decay via a conserved class of stem-loop recognition motifs.

Tumor necrosis factor-α (TNF-α) is the most potent proinflammatory cytokine in mammals. The degradation of TNF-α mRNA is critical for restricting TNF-α synthesis and involves a constitutive decay element (CDE) in the 3' UTR of the mRNA. Here, we demonstrate that the CDE folds into an RNA stem-loop motif that is specifically recognized by Roquin and Roquin2. Binding of Roquin initiates degradation of TNF-α mRNA and limits TNF-α production in macrophages. Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE sequences are highly conserved and are found in more than 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, CDE-containing mRNAs were identified as the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs.