Sharp Judith A, Sparago Emily, Thomas Rachael, Alimenti Kaitlyn, Wang Wei, Blower Michael D
Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, Boston, Massachusetts, United States of America.
PLoS Genet. 2025 Jun 10;21(6):e1011719. doi: 10.1371/journal.pgen.1011719. eCollection 2025 Jun.
SAF-A/HNRNPU is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. Here we show the SAF-A protein is highly dynamic and interacts with nascent transcripts as part of this dynamic movement. This finding revises current models of SAF-A: rather than being part of a static nuclear scaffold/matrix structure that acts as a stable tether between RNA and chromatin, SAF-A executes nuclear functions as a dynamic protein, suggesting contacts between SAF-A, RNA, and chromatin are more high turnover interactions than previously appreciated. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA and RNA. Phosphorylation of SAP domain serines S14 and S26 is important for SAF-A localization and function during mitosis, however, whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain, and SAP domain serines S14 and S26 are required to maintain XIST RNA localization and XIST-dependent histone modifications on the inactive X chromosome, to execute normal protein dynamics, and to maintain normal cell proliferation. In addition, we present evidence that a Xi localization signal resides in the SAP domain, enabling SAF-A to engage with the Xi compartment in a manner distinct from other nuclear territories. We found that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with nuclear structures during interphase. Our data suggest that different nuclear compartments may have distinct requirements for the SAF-A SAP domain to execute nuclear functions, a level of control that was not previously known.
SAF-A/HNRNPU在整个脊椎动物中都保守存在，并已成为调节多种核功能的重要因子，这些功能包括长链非编码RNA（lncRNA）定位、基因表达和剪接。在此我们表明，SAF-A蛋白具有高度动态性，并在这种动态运动过程中与新生转录本相互作用。这一发现修正了当前关于SAF-A的模型：SAF-A并非作为RNA与染色质之间稳定连接的静态核支架/基质结构的一部分，而是作为一种动态蛋白执行核功能，这表明SAF-A、RNA和染色质之间的接触是比之前所认识到的更高周转率的相互作用。SAF-A有几个功能结构域，包括一个直接结合DNA和RNA的N端SAP结构域。SAP结构域丝氨酸S14和S26的磷酸化对于有丝分裂期间SAF-A的定位和功能很重要，然而，这些丝氨酸是否参与SAF-A的间期功能尚不清楚。在本研究中，我们测试了SAP结构域以及SAP结构域丝氨酸S14和S26在X染色体失活、蛋白质动态、基因表达、剪接和细胞增殖中的作用。在此我们表明，SAP结构域以及SAP结构域丝氨酸S14和S26是维持XIST RNA在失活X染色体上的定位以及XIST依赖的组蛋白修饰、执行正常蛋白质动态和维持正常细胞增殖所必需的。此外，我们提供证据表明，一个X染色体失活定位信号存在于SAP结构域中，使SAF-A能够以不同于其他核区域的方式与X染色体失活区域结合。我们发现，维持基因表达不需要SAP结构域，并且它在mRNA剪接中仅起次要作用。我们提出了一个模型，即SAF-A丝氨酸S14和S26的动态磷酸化介导了间期SAF-A与核结构相互作用的快速周转。我们的数据表明，不同的核区域对于SAF-A SAP结构域执行核功能可能有不同的要求，这是一个此前未知的调控水平。
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