The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.
The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.
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DOI：
10.1101/gad.271528.115
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发表时间：
2015-12-15
影响因子：
10.5
通讯作者：
Wahl MC
中科院分区：
文献类型：
--
作者：
Absmeier E;Wollenhaupt J;Mozaffari-Jovin S;Becke C;Lee CT;Preussner M;Heyd F;Urlaub H;Lührmann R;Santos KF;Wahl MC
在这项研究中，Absmeier等人使用X射线晶体学、交联/质谱法以及体内和体外生化功能研究的组合来研究Brr 2解旋酶NTR的结构组织、功能和分子机制。这些发现揭示了防止过早和非生产性三snRNP破坏的分子机制，并提出了Brr 2依赖性剪接的新调控。Brr 2解旋酶为剪接体催化激活提供关键的重塑活性，在此期间它会破坏U4/U6 di-snRNP（小核RNA蛋白），并且其活性必须受到严格调节。Brr 2表现出一种不寻常的结构，包括一个500个残基的N-末端区域，其功能和分子机制目前尚不清楚，其次是一个串联阵列的结构相似的解旋酶单位（盒），只有第一个是催化活性。在这里，我们显示的晶体结构分析的全长Brr 2在复杂的调节Jab 1/MPN结构域的Prp 8蛋白和交联/质谱分离Brr 2的Brr 2的N-末端区域包括两个折叠的结构域和相邻的线性元件，钳和互连的解旋酶盒。逐步的N-末端截短导致酵母生长和剪接缺陷，减少Brr 2与U4/U6·U 5三-snRNP的结合，并增加三-snRNP的ATP依赖性破坏，产生U4/U6二-snRNP和U 5 snRNP。Brr 2截短变体的RNA结合、ATP酶和解旋酶活性的趋势通过晶体结构完全合理化，表明N-末端区域通过底物竞争和构象钳制自抑制Brr 2。我们的研究结果揭示了分子机制，防止过早和非生产性三snRNP中断，并提出新的原则Brr 2依赖性剪接调控。
In this study, Absmeier et al. used a combination of X-ray crystallography, cross-linking/mass spectrometry, and in vivo and in vitro biochemical functional investigations to investigate the structural organization, functions, and molecular mechanisms of the NTR of the Brr2 helicase. The findings reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel regulation of Brr2-dependent splicing. The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.