University of Muenster, Institute for Physical Chemistry, 48149 Muenster, Germany.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA.
RNA. 2020 Nov;26(11):1557-1574. doi: 10.1261/rna.075580.120. Epub 2020 Jul 15.
RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.
RNA 解旋酶催化 ATP 依赖性 RNA 双链体的不稳定。DEAD 框解旋酶共享一个解旋酶核心，介导 ATP 结合和水解、RNA 结合和解旋。该家族的大多数成员包含侧翼核心的结构域，这些结构域可以赋予 RNA 底物特异性，并指导解旋酶到达特定的 RNA。然而，大多数解旋酶的体内 RNA 底物目前尚未确定。来自的 DEAD 框解旋酶 Hera 包含一个解旋酶核心，其后是二聚化结构域和一个 RNA 结合结构域，该结构域折叠成 RNA 识别基序（RRM）。RRM 介导与 RNA 发夹的高亲和力结合，然后由解旋酶核心解开相邻的双链体。Hera 是冷休克蛋白，在冷休克条件下被认为是 RNA 伴侣。使用 Hera/RNA 复合物的交联免疫沉淀和测序，我们表明 Hera 在正常生长和冷休克条件下与大量的 RNA 结合，而没有强烈的序列偏好，这与对 RNA 的结构特异性识别和在 RNA 代谢中的一般功能一致。在冷休克条件下，Hera 被招募到具有高形成稳定二级结构倾向的 RNA 上。我们表明，鉴定出的选定 RNA，包括一组 tRNA，与体外的 Hera 结合，并激活 Hera 解旋酶核心。基因本体分析显示与翻译相关的基因富集，包括核糖体蛋白的 mRNA、tRNA、tRNA 连接酶和 tRNA 修饰酶，这与 Hera 在核糖体和 tRNA 代谢中的关键作用一致。
