Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs.
Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs.
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DOI：
10.1074/jbc.m117.800466
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发表时间：
2017-11-17
期刊：
影响因子：
--
通讯作者：
Rosen MK
中科院分区：
文献类型：
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作者：
Lin Y;Currie SL;Rosen MK
液-液相分离(LLP)被认为有助于建立许多生物分子凝聚物，即浓缩不同大分子但缺乏结合膜的真核细胞结构。RNA颗粒控制着RNA的新陈代谢，并包含一大类凝聚体，富含与RNA结合的蛋白质和RNA分子。许多RNA颗粒蛋白由模块结构域和固有无序区组成，具有较低的氨基酸序列复杂性。这些分子的相分离可能对RNA颗粒的生成和稳定性起着重要作用。为了了解折叠结构域和IDR如何协同调节LLP，我们产生了一系列工程蛋白。这些是基于来自RNA颗粒蛋白FUS(在肉瘤中融合)的IDR与多价Poly-Src Homology 3(SH3)结构域蛋白的融合，当与富含多脯氨酸基序(PolyPRM)的配体混合时，该蛋白会发生相分离。我们发现，野生型IDR促进了PolySH3-PolyPRM体系的LLP，使相分离阈值浓度降低了8倍。IDR的Gly/Ser-Tyr-Gly/Ser基序中酪氨酸残基的系统突变降低了这种影响，这取决于这些替换的数量，而不是这些替换的位置。将所有酪氨酸突变为非芳香族残基或磷酸化IDR使相分离阈值高于未修饰的PolySH3-PolyPRM对。这些结果表明，低复杂性的IDRs可以对LLP进行正向和负向的调节，这取决于芳香性程度和磷酸化状态。我们的发现提供了可信的机制，通过这些机制，这些序列可以改变进化和细胞时间尺度上的RNA颗粒属性。
Liquid–liquid phase separation (LLPS) is thought to contribute to the establishment of many biomolecular condensates, eukaryotic cell structures that concentrate diverse macromolecules but lack a bounding membrane. RNA granules control RNA metabolism and comprise a large class of condensates that are enriched in RNA-binding proteins and RNA molecules. Many RNA granule proteins are composed of both modular domains and intrinsically disordered regions (IDRs) having low amino acid sequence complexity. Phase separation of these molecules likely plays an important role in the generation and stability of RNA granules. To understand how folded domains and IDRs can cooperate to modulate LLPS, we generated a series of engineered proteins. These were based on fusions of an IDR derived from the RNA granule protein FUS (fused in sarcoma) to a multivalent poly-Src homology 3 (SH3) domain protein that phase-separates when mixed with a poly-proline–rich-motif (polyPRM) ligand. We found that the wild-type IDR promotes LLPS of the polySH3–polyPRM system, decreasing the phase separation threshold concentration by 8-fold. Systematic mutation of tyrosine residues in Gly/Ser-Tyr-Gly/Ser motifs of the IDR reduced this effect, depending on the number but not on the position of these substitutions. Mutating all tyrosines to non-aromatic residues or phosphorylating the IDR raised the phase separation threshold above that of the unmodified polySH3–polyPRM pair. These results show that low-complexity IDRs can modulate LLPS both positively and negatively, depending on the degree of aromaticity and phosphorylation status. Our findings provide plausible mechanisms by which these sequences could alter RNA granule properties on evolutionary and cellular timescales.