Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), 50931 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, Institute of Genetics, University of Cologne, 50931 Cologne, Germany and Leibniz-Institute of Freshwater Ecology and Inland Fisheries, IGB, Müggelseedamm 301, 12587 Berlin, Germany.
Nucleic Acids Res. 2014 Mar;42(5):3177-93. doi: 10.1093/nar/gkt1311. Epub 2013 Dec 17.
NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.
NKAP 是一种高度保守的蛋白质，在转录抑制、T 细胞发育、成熟以及获得功能能力以及维持和存活成人造血干细胞中发挥作用。在这里，我们报告了 NKAP 在剪接中的新作用。使用 NKAP 特异性抗体，我们发现 NKAP 定位于核斑点。NKAP 在 N 端具有 RS 基序，其后是碱性结构域和 C 端区域的 DUF926 结构域。缺失分析表明碱性结构域对于斑点定位很重要。在下拉实验中，我们鉴定了 RNA 结合蛋白、RNA 解旋酶和剪接因子作为 NKAP 的相互作用伙伴，其中包括 FUS/TLS。FUS/TLS-NKAP 相互作用通过 NKAP 的 RS 结构域和 FUS/TLS 的 RGG1 和 RGG3 结构域发生。我们使用体外剪接实验分析了 NKAP 与 RNA 相互作用的能力，发现 NKAP 结合了剪接的信使 RNA（mRNA）和未剪接的前体 RNA。使用交联和免疫沉淀-seq 的全基因组分析显示 NKAP 与 U1、U4 和 U5 小核 RNA 相关联，我们还证明了 NKAP 的敲低导致前体 RNA 百分比增加。我们的结果揭示了 NKAP 作为具有 RNA 剪接和加工作用的核斑点蛋白。
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