RC3H1 posttranscriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kB pathway [RNA-Seq]

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFalpha mRNA decay via a 3''UTR constitutive decay element (CDE). Here, we applied PAR-CLIP to human RC3H1 to identify about 3800 mRNA targets with more than 16000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage induced mRNAs, indicating a role of this RNA-binding protein in the posttranscriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of NF-kB pathway regulators such as IkBalpha and A20. RC3H1 uses roquin and Zn-finger domains to contact a binding site in the A20 3''UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IkB kinase and NF-kB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-kB pathway. Overall design: We measured global mRNA decay rates in mock and RC3H1/RC3H2-depleted HEK293 cells. Transcription was blocked by Actinomycin D zero, one or two hours before harvesting. Total RNA was isolated in two biological replicates and subjected to polyA selection followed by high-throughput sequencing.

RNA结合蛋白（RNA-binding protein）RC3H1（又称ROQUIN）可通过3'非翻译区（3''UTR）的组成型降解元件（CDE）促进肿瘤坏死因子α（TNFα）mRNA的降解。本研究采用PAR-CLIP技术对人源RC3H1进行实验，共鉴定出约3800个mRNA靶标，涵盖16000余个结合位点。其中大量结合位点与共识型CDE存在差异，并揭示出一种结构-序列基序：富含尿嘧啶（U）的序列嵌入发夹结构内部。RC3H1优先结合半衰期较短的mRNA以及DNA损伤诱导产生的mRNA，提示该RNA结合蛋白在DNA损伤应答的转录后调控中发挥重要功能。值得注意的是，RC3H1可调控核因子κB（NF-κB）通路调控因子的表达，例如IκBα与A20。RC3H1通过Roquin结构域与锌指结构域（Zn-finger domains）与A20的3''UTR区域内的结合位点发生相互作用，揭示了一种此前未被报道的RC3H1结合模式。RC3H1基因敲低可导致A20蛋白表达水平上调，进而干扰IκB激酶（IKK）与NF-κB的活性，证实RC3H1能够调控IKK/NF-κB通路的活性。实验整体设计：本研究在空白对照（mock）及RC3H1/RC3H2双敲低的HEK293细胞中检测全基因组mRNA的降解速率。在收获细胞前0、1或2小时，采用放线菌素D（Actinomycin D）阻断转录过程。通过两次生物学重复提取总RNA，随后进行polyA富集并开展高通量测序。