Decoding and recoding phase behavior of TDP43 reveals that phase separation is not required for splicing function
Decoding and recoding phase behavior of TDP43 reveals that phase separation is not required for splicing function
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DOI：
10.1101/548339
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发表时间：
2019-02
期刊：
影响因子：
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通讯作者：
H. B. Schmidt;A. Barreau;R. Rohatgi
中科院分区：
文献类型：
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作者：
H. B. Schmidt;A. Barreau;R. Rohatgi
内含子无序区（IDR）通常是快速进化的低序列复杂性蛋白质结构域，可以驱动相变，通常存在于许多与神经退行性疾病相关的蛋白质中，包括RNA加工因子TDP43。然而，相分离如何有助于细胞中TDP43的生理功能仍然是个谜。我们结合了系统的诱变指导下的进化序列分析与活细胞的TDP43相动力学的报告分析，以确定定期间隔的疏水基序分离的灵活，亲水性的IDR段作为TDP43相特性的关键决定因素。这种启发式框架允许我们定制TDP43缩合物的材料性质，以确定对剪接功能的影响。值得注意的是，具有增加或减少的相动力学的突变体，甚至不能相分离的突变体，可以介导定量的单细胞剪接报告基因和内源性靶的剪接。我们的结论是，相分离是不需要的TDP43的剪接功能。
Intrinsically disordered regions (IDRs) often are fast evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. We combined systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allowed us to customize the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, mutants with increased or decreased phase dynamics, and even mutants that failed to phase separate, could mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. We conclude that phase separation is not required for the function of TDP43 in splicing.