A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation.
A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation.
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DOI：
10.3389/fmolb.2021.691694
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发表时间：
2021
影响因子：
5
通讯作者：
Bah A
中科院分区：
文献类型：
--
作者：
Dettori LG;Torrejon D;Chakraborty A;Dutta A;Mohamed M;Papp C;Kuznetsov VA;Sung P;Feng W;Bah A
R环是由DNA：RNA杂合体、置换的单链（ss）DNA和尾随的ssRNA突出端组成的非规范的三链核酸结构。R环在正常和疾病条件下都具有重要的生物学功能。为了阐明它们的细胞功能，我们需要了解R环形成、识别、信号传导和分解的机制。先前的高通量筛选鉴定了结合R环的多种蛋白质，其中许多这些蛋白质含有防止（例如，拓扑异构酶），解析（例如，解旋酶，核酸酶），或识别（例如，KH、RRM）R环。然而，大量的这些R环相互作用的酶和阅读器蛋白还含有长的固有无序区（IDR）。折叠结构域和IDR协同识别和处理R环或调节R环介导的信号传导的精确分子和结构机制尚未完全探索。在研究一种这样的模块化R环阅读器，脆性X蛋白（FMRP）时，我们意外地发现FMRP的C末端IDR（C-IDR）是主要的R环结合位点，三个N末端KH结构域识别尾随的ssRNA突出端。有趣的是，FMRP的C-IDR最近已被证明可以通过自身或与另一种非经典核酸结构RNA G-四链体复合进行自发的液-液相分离（LLPS）组装。此外，我们最近发现FMRP可以抑制转录过程中形成的持久性R环，这一过程也通过LLPS通过组装无膜转录工厂而得到增强。这些令人兴奋的发现促使我们通过全面的生物信息学和计算生物学研究来探索IDR在R环加工和信号蛋白中的作用。在这里，我们评估了IDR患病率，序列组成和LLPS倾向的已知R环相互作用组。我们观察到，像FMRP一样，大多数R环相互作用组，特别是Readers，含有高度富集在具有偏向氨基酸组成的低复杂性序列中的长IDR，表明这些IDR可以直接与R环相互作用，而不是连接"功能性折叠酶或结合结构域"的"仅仅是柔性接头"。此外，我们的分析表明，在R-环相互作用组中的几种蛋白质被预测或已被实验证明进行LLPS或已知与相分离的无膜细胞器。因此，我们的总体结果提出了一个发人深省的假设，即R环相互作用组中的IDR可以通过直接结合和下游信号传导通过LLPS介导的无膜R环焦点的组装提供R环识别之间的功能联系。IDR富集的R环相互作用物的功能的缺失或失调可能导致严重的基因组缺陷，例如我们最近在脆性X患者来源的细胞中观察到的广泛的R环介导的DNA双链断裂。
R-loops are non-canonical, three-stranded nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under both normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, with many of these proteins containing folded nucleic acid processing and binding domains that prevent (e.g., topoisomerases), resolve (e.g., helicases, nucleases), or recognize (e.g., KH, RRMs) R-loops. However, a significant number of these R-loop interacting Enzyme and Reader proteins also contain long stretches of intrinsically disordered regions (IDRs). The precise molecular and structural mechanisms by which the folded domains and IDRs synergize to recognize and process R-loops or modulate R-loop-mediated signaling have not been fully explored. While studying one such modular R-loop Reader, the Fragile X Protein (FMRP), we unexpectedly discovered that the C-terminal IDR (C-IDR) of FMRP is the predominant R-loop binding site, with the three N-terminal KH domains recognizing the trailing ssRNA overhang. Interestingly, the C-IDR of FMRP has recently been shown to undergo spontaneous Liquid-Liquid Phase Separation (LLPS) assembly by itself or in complex with another non-canonical nucleic acid structure, RNA G-quadruplex. Furthermore, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to explore the role of IDRs in R-loop processing and signaling proteins through a comprehensive bioinformatics and computational biology study. Here, we evaluated IDR prevalence, sequence composition and LLPS propensity for the known R-loop interactome. We observed that, like FMRP, the majority of the R-loop interactome, especially Readers, contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition, suggesting that these IDRs could directly interact with R-loops, rather than being “mere flexible linkers” connecting the “functional folded enzyme or binding domains”. Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Thus, our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome can provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci. The absence or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the widespread R-loop-mediated DNA double strand breaks that we recently observed in Fragile X patient-derived cells.