Skrajna Aleksandra, Yang Xiao-Cui, Tarnowski Krzysztof, Fituch Kinga, Marzluff William F, Dominski Zbigniew, Dadlez Michał
Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
J Mol Biol. 2016 Mar 27;428(6):1180-1196. doi: 10.1016/j.jmb.2016.01.031. Epub 2016 Feb 6.
Histone pre-mRNAs are cleaved at the 3' end by a complex that contains U7 snRNP, the FLICE-associated huge protein (FLASH) and histone pre-mRNA cleavage complex (HCC) consisting of several polyadenylation factors. Within the complex, the N terminus of FLASH interacts with the N terminus of the U7 snRNP protein Lsm11, and together they recruit the HCC. FLASH through its distant C terminus independently interacts with the C-terminal SANT/Myb-like domain of nuclear protein, ataxia-telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both hydrogen/deuterium exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.
组蛋白前体信使核糖核酸（pre - mRNA）在3'端被一个复合物切割，该复合物包含U7小核核糖核蛋白（snRNP）、FLICE相关巨蛋白（FLASH）以及由几种聚腺苷酸化因子组成的组蛋白前体mRNA切割复合物（HCC）。在该复合物中，FLASH的N端与U7 snRNP蛋白Lsm11的N端相互作用，它们共同招募HCC。FLASH通过其较远的C端独立地与核蛋白共济失调毛细血管扩张症基因座（NPAT）的C端SANT/Myb样结构域相互作用，NPAT是S期组蛋白基因表达所需的转录共激活因子。为了获得这些相互作用的结构信息，我们使用质谱法监测FLASH、Lsm11和NPAT单独或在其各自结合伴侣存在时不同区域的氢/氘交换。我们的结果表明，Lsm11中与FLASH相互作用的结构域高度动态，而招募HCC所需的更下游区域氘交换缓慢，可能折叠成稳定结构。在FLASH中，与Lsm11相互作用的结构域采用稳定结构，并且该结构域通过结合Lsm11进一步稳定。值得注意的是，氢/氘交换实验和体外结合试验均表明，Lsm11除了与FLASH的N端区域相互作用外，还与FLASH的C端SANT/Myb样结构域接触，该区域也是与NPAT结合的区域。然而，虽然NPAT使该结构域稳定，但Lsm11导致其部分松弛。这些相互竞争的反应可能在体内调节组蛋白基因表达中起作用。
