RNA Affinity Purification and Characterization of Roquin Proteins in CDE-mediated mRNA Decay

Tumor necrosis factor (TNF)-α is the most potent pro-inflammatory cytokine in mammals. The degradation of TNFα mRNA is critical for restricting TNFα synthesis and involves an AU-rich element (ARE) and a constitutive decay element (CDE) in the 3' untranslated region (UTR) of the mRNA. In the first part of my thesis, I optimized an RNA-based method to identify RNA-binding proteins (BPs) associated with TNFα mRNA. For this, I developed a modified streptavidin-binding RNA aptamer termed S1m. It has improved affinity for streptavidin and I found a four-fold repeat (4xS1m) to be most efficient. I then used TNFα ARE-4xS1m RNA to purify ARE-BPs from cellular extracts. By this, I found the majority of established ARE-BPs and confirmed Rbms1 and Roxan as novel ARE-BPs. The optimized 4xS1m aptamer therefore provides a powerful tool for the discovery of ribonucleoprotein (RNP) components. In the second part of my thesis, I investigated the TNFα CDE in detail and found that the CDE is a 17 nucleotide long structured motif. Structural probing and mutagenesis provide evidence that it folds into a short RNA stem-loop in its active conformation. Using my 4xS1m protocol, I then identified CDE-associated proteins by mass spectrometry. Thereby, I found that the CCCH-type zinc and RING finger proteins Roquin (Rc3h1) and its paralog Roquin2 (Rc3h2) are stem-loop specific CDE-BPs. Next, I confirmed that the ROQ domain of Roquin specifically and directly binds to the CDE stem-loop. I could further show that Roquin is required for CDE-mediated mRNA decay and suppression of TNFα production in macrophages. TNFα expression was also increased by introduction of a morpholino that interferes with CDE-Roquin binding. My data provide evidence that Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE motifs are highly conserved and are found in over 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, I confirmed that CDE-containing mRNAs are 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. In all, I unraveled a mechanism that adds an important component to the complex network that governs posttranscriptional control of gene expression

Genome-Wide Assessment of AU-Rich Elements by the AREScore Algorithm

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila

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

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

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

SummaryThe 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

Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5′ UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, “humanized” yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators. © 2020 Elsevier Inc.ISSN:1097-2765ISSN:1097-416

ARE<i>Score</i> distribution in comparison to randomized controls.

<p>(A) The ARE<i>Score</i> distribution of the <i>H. sapiens</i> transcriptome (every annotated transcript with a 3′UTR length ≥10 nt) was compared to a fully adjusted, randomized control set of sequences with identical lengths and A/T/G/C-content. Percentage of transcripts is depicted on a logarithmic scale. (B) The same analysis was done with the <i>D. melanogaster</i> transcriptome, as in panel A. Frequencies of 0 were omitted from the graph. (C) The same analysis was done with the <i>C. elegans</i> transcriptome, as in panel B. (D) The frequency of mRNAs with an ARE<i>Score</i> ≥10 in the actual transcriptome of 14 species was compared to the frequency in fully adjusted, randomized control sequences. The analysis was carried out for <i>Amphimedon queenslandica</i> (demosponge), <i>Hydra magnipapillata</i> (freshwater polyp), <i>Aplysia californica</i> (California sea hare), <i>Caenorhabditis elegans</i> (roundworm), <i>Ixodes scapularis</i> (deer tick), <i>Drosophila melanogaster</i> (fruit fly), <i>Strongylocentrotus purpuratus</i> (purple sea urchin), <i>Ciona intestinalis</i> (vase tunicate), <i>Branchiostoma floridae</i> (Florida lancelet), <i>Danio rerio</i> (zebrafish), <i>Xenopus laevis</i> (African clawed frog), <i>Gallus gallus</i> (chicken), <i>Mus musculus</i> (common house mouse) and <i>Homo sapiens</i> (man). The Φ coefficient serves as a measure for how strongly ARE<i>Scores</i> ≥10 are associated with the actual transcriptome as compared to the randomized control. P-values were calculated by χ²-test, n represents the number of transcripts. Species labeled in red show a significant enrichment of mRNAs with ARE<i>Scores</i> ≥10.</p

Analysis of Tis11-sensitive mRNAs in <i>D. melanogaster</i> SL2 cells.

<p>(A) <i>D. melanogaster</i> SL2 cells were treated over a period of 4 days with 12.5 µg/ml dsRNA in order to knock down Tis11, or with dsRNA targeting GFP as a control. Total RNA was extracted from three biological repeats for microarray analysis using the Affymetrix Drosophila Genome 2.0 array. After normalization of the signal intensities using Robust Multi-array Analysis (RMA), the fold change of expression by Tis11 kd (signal in Tis11 kd/signal in GFP kd) was calculated. The list shows all 53 mRNAs with a log2-transformed fold change of >0.5, i.e. a fold change of >1.41. Statistical significance was determined by rank products (RP) test and independently by Student's T-test (TT). A heat map of the signal intensities in the three biological repeats is provided on the left side, the ARE<i>Score</i> is shown on the right side. (B) The ARE<i>Score</i> distribution is depicted for 49 out of the 53 Tis11-sensitive mRNAs identified in panel A. Only mRNAs with an annotated 3′UTR length ≥10 nt were included. The ARE<i>Score</i> distribution of the entire <i>D. melanogaster</i> transcriptome serves as the control group.</p