Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, AB, T1K 3M4, Canada.
Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada.
Nucleic Acids Res. 2021 Nov 18;49(20):11810-11822. doi: 10.1093/nar/gkab934.
The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure-function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function.
人类假尿嘧啶核苷合成酶 PUS7 是一种多功能的 RNA 修饰酶，能够靶向许多 RNA，从而在发育和大脑功能中发挥关键作用。虽然 PUS7 的所有靶 RNA 都具有保守序列，但可能需要额外的识别元件，并且 PUS7 与 RNA 结合的结构基础尚不清楚。在这里，我们描述了人源 PUS7 的结构-功能关系，并报告了其 2.26Å分辨率的 X 射线晶体结构。与细菌同源物相比，人源 PUS7 具有两个额外的亚结构域，结构建模研究表明这些亚结构域通过与 tRNA 底物的增加相互作用有助于 tRNA 的识别。与我们的建模一致，我们发现 PUS7 与 tRNA 的所有结构元件都需要进行有效的相互作用，因为靶 RNA 的保守序列本身不足以进行人源 PUS7 的假尿嘧啶化。此外，PUS7 以中等亲和力结合几种不可修饰的 RNA，这可能使 PUS7 能够筛选出有活性的 RNA 底物。在 tRNA 修饰后，产物 tRNA 与 PUS7 的亲和力显著降低，从而促进其解离。总之，我们的研究表明 PUS7 对 RNA 的识别具有结构特异性和序列特异性，并为其功能提供了机制上的见解。
