SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD is a recently characterized, abundant, and highly conserved cytoplasmic lncRNA that is required for proper mitotic divisions in human cells. NORAD antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. The interactions between NORAD, Pumilio and SAM68 provide a paradigm for how specific repeated and structured elements with a lncRNA can facilitate its function. Overall design: RNA-seq following perturbations of SAM68 in U2OS cells and NORAD lncRNA in HCT116 WT and SAM68 KO cells

已知长链非编码RNA（long noncoding RNA，lncRNA）的功能报道数量正快速增长，但这些功能如何在其序列与结构中被编码，目前仍缺乏深入认知。NORAD是近期被鉴定与表征的、丰度较高且高度保守的细胞质lncRNA，对人类细胞的正常有丝分裂至关重要。NORAD能够拮抗Pumilio家族的阻遏蛋白，该家族蛋白可结合NORAD序列中12个重复单元所分布的至少17个结合位点。本研究对NORAD重复序列中的保守区域展开分析，鉴定出更多的互作伴侣蛋白，并解析了NORAD与RNA结合蛋白SAM68（KHDRBS1）之间的相互作用——该互作是NORAD发挥拮抗Pumilio蛋白功能所必需的。NORAD、Pumilio与SAM68之间的相互作用，为长链非编码RNA内特定的重复与结构化元件如何助力其功能实现提供了研究范式。整体实验设计：在U2OS细胞中对SAM68进行扰动处理，以及在HCT116野生型（WT）与SAM68基因敲除（KO）细胞中对NORAD lncRNA进行扰动处理后开展RNA测序（RNA-seq）。