Capturing the hierarchically assorted modules of protein-protein interactions in the organized

Nuclear proteins are major constituents and key regulators of nucleome topological organization and ma-nipulators of nuclear events. To decipher the global connectivity of nuclear proteins and the hierarchically organized modules of their interactions, we conducted two rounds of cross-linking mass spectrometry (XL -MS) analysis, one of which followed a quantitative double chemical cross-linking mass spectrometry (in vivo qXL-MS) workflow, and identified 24,140 unique crosslinks in total from the nuclei of soybean seed-lings. This in vivo quantitative interactomics enabled the identification of 5340 crosslinks that can be con-verted into 1297 nuclear protein-protein interactions (PPIs), 1220 (94%) of which were non-confirmative (or novel) nuclear PPIs compared with those in repositories. There were 250 and 26 novel interactors of his-tones and the nucleolar box C/D small nucleolar ribonucleoprotein complex, respectively. Modulomic anal-ysis of orthologous Arabidopsis PPIs produced 27 and 24 master nuclear PPI modules (NPIMs) that contain the condensate-forming protein(s) and the intrinsically disordered region-containing proteins, respec-tively. These NPIMs successfully captured previously reported nuclear protein complexes and nuclear bodies in the nucleus. Surprisingly, these NPIMs were hierarchically assorted into four higher-order com-munities in a nucleomic graph, including genome and nucleolus communities. This combinatorial pipeline of 4C quantitative interactomics and PPI network modularization revealed 17 ethylene-specific module var-iants that participate in a broad range of nuclear events. The pipeline was able to capture both nuclear pro-tein complexes and nuclear bodies, construct the topological architectures of PPI modules and module variants in the nucleome, and probably map the protein compositions of biomolecular condensates.

Automated summary

In this study, the researchers used cross-linking mass spectrometry to analyze the nuclear proteins in soybean seedlings, and identified 1297 nuclear protein-protein interactions. They also constructed a network model for these interactions, and discovered several ethylene-specific module variants.