thiol(SH)-linked alkylation for the metabolic sequencing (SLAM-seq) of RNA after Crm1 inhibition and Nup1/Nup2 degradation

Nuclear pore proteins (Nups) physically interact with hundreds of chromosomal sites, impacting transcription. In yeast, transcription factors mediate interactions between Nups and enhancers and promoters. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC). This mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 and Nups co-occupy enhancers and Crm1 inhibition blocks interaction of the nuclear pore protein Nup2 with the genome. In vivo, Crm1 interacts stably with the NPC. In vitro, Crm1 binds both Gcn4 and Nup2 directly. Importantly, the interaction between Crm1 and Gcn4 requires neither Ran-GTP nor the nuclear export sequence binding site. Finally, Crm1 and Ran-GTP stimulate DNA binding by Gcn4, suggesting that allosteric coupling between Crm1-Ran-GTP binding and DNA binding facilitates docking of transcription factor-bound enhancers at the NPC.核孔蛋白（Nuclear pore proteins, Nups）可与数百个染色体位点发生物理相互作用，进而调控转录过程。在酵母中，转录因子可介导核孔蛋白与增强子、启动子之间的相互作用。为阐明该分子机制的本质，本研究利用了Gcn4转录因子的功能分离突变体，该突变可阻断其与核孔复合体（Nuclear pore complex, NPC）的结合。该突变同时会减弱Gcn4与高度保守的核输出因子Crm1/Xpo1之间的相互作用。Crm1与核孔蛋白可共同占据增强子区域，且抑制Crm1能够阻断核孔蛋白Nup2与基因组的结合。体内实验显示，Crm1可与核孔复合体稳定结合；体外实验则证实，Crm1可直接结合Gcn4与Nup2二者。值得注意的是，Crm1与Gcn4之间的相互作用既不依赖Ran-GTP，也不需要核输出序列结合位点。最后，Crm1与Ran-GTP可促进Gcn4的DNA结合能力，这提示Crm1-Ran-GTP结合与DNA结合之间的别构耦合，能够助力结合了转录因子的增强子在核孔复合体处的锚定。