Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2
Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2
复制标题
DOI：
10.1080/15384101.2016.1255384
复制
发表时间：
2017-01-01
期刊：
影响因子：
4.3
通讯作者：
Wahl, Markus C.
中科院分区：
文献类型：
--
作者：
Absmeier, Eva;Becke, Christian;Wahl, Markus C.
RNA解旋酶Brr2与前mRNA剪接的多个阶段有关，因此需要严格的调控。Brr2可以通过一个大的N-末端区域折叠回到其解旋酶核心而被自动抑制，并被一个催化失活的C-末端解旋酶盒自动激活。此外，它还可以受Prp8蛋白的Jab1结构域的反式调节，该结构域可以通过在酶的RNA结合隧道中间歇插入C末端的尾巴来抑制Brr2，或者在去掉这条尾巴后激活解旋酶。目前尚不清楚这些调控机制是否在功能上相互作用，以及它们在进化上的保守程度。在这里，我们报道了酿酒酵母和嗜热毛壳菌Brr2-Jab1复合体的晶体结构，证明了基于Jab1的Brr2抑制可能在所有真核生物中都有效，但是通过生物特异性的分子接触实现的。此外，结构表明Brr2的自动抑制可以与Jab1介导的抑制协同作用，并表明N-末端区域影响Jab1 C-末端尾部在RNA结合隧道中的相互作用。系统的RNA结合和解离研究表明，N-末端区域和Jab1 C-末端尾部分别特异性地干扰RNA底物的双链和单链区域的调节，相互促进。此外，这些分析表明，基于N-末端区域的调控需要存在不活跃的C-末端解旋酶盒。综上所述，我们的结果概述了一个复杂的调控机制系统，这些机制在SnRNP组装和剪接过程中控制Brr2的活动。
RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.