Iowa State University, Biomedical Sciences, Ames, IA, USA.
Nucleic Acids Res. 2018 Nov 16;46(20):10983-11001. doi: 10.1093/nar/gky770.
The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.
存活运动神经元 (SMN) 蛋白对于所有动物细胞的存活都是必不可少的。SMN 具有核酸结合结构域，在 RNA 代谢中发挥着重要作用。然而，SMN 的 RNA 结合特性还不太清楚。在这里，我们采用迭代体外选择和化学结构探测来鉴定与 RNA-SMN 相互作用相关的序列和结构基序。我们的结果表明，驱动 RNA-SMN 相互作用的基序是多种多样的，并表明紧密的 RNA-SMN 相互作用需要 RNA 分子上存在多个接触位点。我们进行了紫外线交联和免疫沉淀与高通量测序 (HITS-CLIP)，以鉴定神经元 SH-SY5Y 细胞中 SMN 的细胞内 RNA 靶标。HITS-CLIP 的结果鉴定了各种各样的靶标，包括编码核糖体生物发生和细胞骨架动力学的 mRNA。我们在体外显示了 ANXA2 mRNA 与 SMN 直接相互作用的关键决定因素。我们的数据证实了 SMN 区分紧密 RNA 序列的能力，并且代表了 SMN 与细胞内 RNA 靶标直接相互作用的首次验证。我们的发现表明，直接的 RNA-SMN 相互作用是引发导致执行 SMN 特异性功能的级联事件的新机制。
