Mammalian nuclear speckles exhibit stable association with chromatin: a biochemical study

Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a “putative stable association” whose coupling might be subject to the combined regulation from both chromatin and NS changes. <b>Abbreviations:</b> NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.

核斑点（Nuclear Speckles，NS）是一类由蛋白质与RNA组成的相分离冷凝物，其组分可动态调控RNA的转录、剪接、转运以及DNA修复过程。长期以来，这类结构主要通过成像技术开展研究，曾以染色质间颗粒簇（Interchromatin Granule Clusters，IGCs）之名被广泛认知，但其生化特性，尤其是与染色质（Chromatin）的相互作用，在很大程度上仍未得到充分探索。

本研究旨在探究核斑点是否与染色质存在稳定相互作用，结果显示，对染色质进行有限的脱氧核糖核酸酶1（DNase 1）切口处理后，核斑点会解体为各向同性分布的组分或组成蛋白聚集体，且该过程不会影响其他细胞核结构。进一步的生化探究结果表明，核斑点蛋白与染色质紧密结合，仅能通过高盐处理进行提取，这一点与组蛋白（histone proteins）一致。此外，经微球菌核酸酶（Micrococcal Nuclease，MNase）消化染色质后，核斑点可与可溶性单-双核小体染色质组分共同释放。我们提出了一个模型，认为核斑点与染色质构成一种"假定的稳定相互作用"，二者的耦合可能同时受到染色质与核斑点自身变化的共同调控。

<b>缩写说明：</b> NS：核斑点（Nuclear Speckles）；DSB：双链断裂（double strand breaks）；PTM：翻译后修饰（posttranslational modifications）；DDR：DNA损伤修复（DNA damage repair）；RBP：RNA结合蛋白（RNA Binding Proteins）；TAD：拓扑关联结构域（topologically associated domains）；LCR：低复杂度区域（low complexity regions）；IDR：内在无序区域（intrinsically disordered regions）。