Fig. 5: Nsp1 engages with the CTN and cytoplasmic filament subcomplexes in two opposing assembly pathways.
From: Co-translational assembly orchestrates competing biogenesis pathways
a Structure of Chaetomium thermophilum CTN shows the heterotrimeric coiled-coil which is tethered to Nic96 (PDB: 5CWS)16. b RIP-qPCR of the CTN suggests co-translational interactions of Nsp1 with nascent Nup57 but not Nup49. Bar graphs show mean ± SD. n = 5 biologically independent samples for Nsp1-StepII (nsp1- and nup49-mRNA), Nup49-StepII (nsp1[cycloheximide]-, nup49[cycloheximide]- and nup57-mRNA) and Nup57-StepII (nsp1-, nup49-, and nup57-mRNA) and n = 4 biologically independent samples for Nsp1-StepII (nup57-mRNA) and Nup49-StepII (nsp1- and nup49-mRNA under puromycin). **p = 0.0042 for Nsp1-StepII (nup57-mRNA). c Selective ribosome profiling from Nsp1-IPs identifies its co-translational association with nascent Nup57 at the coiled-coil segment 1 (CCS1). Selective ribosome profiling data was generated from n = 4 biologically independent samples. d Structural model of the coiled-coil in the Nup159-subcomplex21. e RIP-qPCR experiments targeting the Nup159-subcomplex (affinity purified Nsp1, Nup82 and Nup159). Nup159 co-translationally binds to nascent Nup82. RIP-qPCR experiments depicted as mean ± SD. n = 6 biologically independent samples for Nsp1-StepII (nup82-mRNA under cycloheximide); n = 5 biologically independent samples for Nsp1-StepII (nup82[puromycin]- and nup159[cycloheximide]-mRNA), Nup82-StepII (nsp1-, nup82- and nup116-mRNA) and Nup159-StepII (nsp1-, nup82- and nup116-mRNA), n = 4 biologically independent samples for Nsp1-StepII (nup159-mRNA under puromycin) and Nup82-StepII (nup159-mRNA) and n = 2 biologically independent samples for Nsp1-StepII (nsp1-mRNA). *p = 0.0465 for Nsp1-StepII (nup82-mRNA) and **p = 0.0015 for Nup159(nup82-mRNA). f SeRP with affinity purified Nsp1 does not detect co-translational association within the Nup159-subcomplex. Selective ribosome profiling was performed with four biologically independent replicates. g Assembly scheme for the CTN- and Nup159-subcomplexes. Nsp1 co-translationally seeds the assembly in the CTN-subcomplex but post-translationally completes the assembly of the Nup159-subcomplex. ns p > 0.05, *p < 0.05, **p < 0.01 (Two-sided, paired t-test). Source data for RIP-qPCR in panel b, and e, are provided as a Source Data file. IP immunoprecipitation, AA amino acid, FG phenylalanine-glycine repeats, cta co-translational assembly, pta: post-translational assembly.
