Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.
Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.
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相分离rna结合蛋白在亚饱和溶液中形成不均匀分布的簇。
DOI：
10.1073/pnas.2202222119
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发表时间：
2022-07-12
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
作者：
无膜生物分子缩合物是具有不同组成偏好和功能的分子群落。人们相当多的注意力集中在宏观相分离作为产生冷凝物的过程。这一焦点隐含地假设共存的稀相和次饱和溶液是无特征的。在这里，我们证明事实并非如此。令人惊讶的是，RNA 结合蛋白的 FUS-EWSR1-TAF15 家族在亚饱和溶液中形成簇的重尾分布。鉴于相分离蛋白的内源浓度较低，我们在亚饱和条件下发现的簇将存在于细胞环境中是理所当然的。了解此类团簇的存在，为评估在不饱和溶液中形成或与冷凝物共存的不同尺寸和形状的团簇的功能相关性打开了大门。大分子相分离被认为是驱动细胞内无膜生物分子凝聚物形成的过程之一。相分离的动力学被认为遵循经典成核理论的原则，因此，次饱和溶液应该不含具有多个分子的簇。我们使用体外生物物理研究测试了这一预测，以表征具有本质无序的朊病毒样结构域和 RNA 结合结构域的相分离 RNA 结合蛋白的亚饱和溶液。令人惊讶的是，与经典成核理论的预期直接矛盾的是，我们发现次饱和溶液的特征是存在团簇的异质分布。随着蛋白质浓度增加并接近饱和浓度，以小物种为主的簇大小的分布不断向更大的大小转变。因此，许多簇包含数十到数百个分子，而不到 1% 的溶液是直径为数百纳米的中尺度物质。我们发现，过饱和溶液中的簇形成和过饱和溶液中的相分离通过序列编码的相互作用紧密耦合。我们还发现，可以使用溶质以及特定的突变组来解耦簇形成和相分离。我们的研究结果与缔合聚合物的预测一致，暗示序列特异性和溶解度决定相互作用网络之间的相互作用，它们分别控制亚饱和溶液中簇的形成和发生相分离的饱和浓度。
Membraneless biomolecular condensates are molecular communities with distinct compositional preferences and functions. Considerable attention has focused on macroscopic phase separation as the process that gives rise to condensates. This focus implicitly presumes that coexisting dilute phases and subsaturated solutions are featureless. Here, we show that this is not the case. Surprisingly, the FUS-EWSR1-TAF15 family of RNA-binding proteins form heavy-tailed distributions of clusters in subsaturated solutions. Given the low endogenous concentrations of phase-separating proteins, it stands to reason that the clusters we have discovered in subsaturated conditions will be present in cellular milieus. Knowing that such clusters exist opens the door to assessing the functional relevance of clusters of different sizes and shapes that form in subsaturated solutions or coexist with condensates. Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.
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影响因子：
17.1
作者：
Arosio, Paolo;Mueller, Thomas;Knowles, Tuomas P. J.
通讯作者：
Knowles, Tuomas P. J.
DOI：
10.1073/pnas.1509317112
发表时间：
2015-09-22
影响因子：
11.1
作者：
Berry, Joel;Weber, Stephanie C.;Brangwynne, Clifford P.
通讯作者：
Brangwynne, Clifford P.
DOI：
10.1073/pnas.1706197114
发表时间：
2017-09-26
影响因子：
11.1
作者：
Brady, Jacob P.;Farber, Patrick J.;Kay, Lewis E.
通讯作者：
Kay, Lewis E.
影响因子：
4.3
作者：
Choi, Jeong-Mo;Dar, Furqan;Pappu, Rohit, V
通讯作者：
Pappu, Rohit, V
影响因子：
5.6
作者：
Alberti S;Saha S;Woodruff JB;Franzmann TM;Wang J;Hyman AA
通讯作者：
Hyman AA