Department of Microbiology, Immunobiology and Genetics, Max Perutz Labs, University of Vienna, Vienna Biocenter (VBC), 1030 Vienna, Austria.
Center for Anatomy and Cell Biology, Medical University of Vienna, 1090 Vienna, Austria.
RNA. 2020 Sep;26(9):1198-1215. doi: 10.1261/rna.074047.119. Epub 2020 May 18.
In , endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.
在原核生物中，内切核酸酶 RNase E 启动许多 RNA 的降解，并作为转录后调控的中心。四聚体衔接蛋白 RapZ 将小分子调节 RNA GlmZ 靶向到 RNase E 的降解。RapZ 通过位于羧基末端的一个结构域结合 GlmZ，并与 RNase E 相互作用，促进 GlmZ 在碱基配对区域的切割。当需要时，同源小分子 RNA GlmY 通过分子模拟来拮抗 GlmZ 的切割，从而解除对 RapZ 的结合。在当前的研究中，我们解决了 RapZ 采用的分子机制。我们表明，在 RNA 结合方面有缺陷但在与 RNase E 结合方面有功能的 RapZ 突变体在增加浓度时能够在体内和体外刺激 GlmZ 的切割。相比之下，保留 RNA 结合活性但无法接触 RNase E 的截断 RapZ 变体则缺乏这种活性。一致地，我们发现四聚体 RapZ 通过与 RNase E 的大球形结构域直接相互作用，独立于 RNA 结合同样四聚体的 RNase E，尽管 RapZ 在体外刺激至少一种非同源 RNA 的切割，但它在体内的活性仅限于 GlmZ，这如 RNA 测序所揭示的那样，表明 RNA 底物中的某些特征也是切割所必需的。总之，RapZ 通过与其催化结构域相互作用来增强 RNase E 的活性，这代表了一种新的 RNase E 激活机制。相比之下，RNA 结合具有招募作用，增加了形成有活性的 RapZ/GlmZ/RNase E 复合物的可能性。
