Protein methylation is emerging as an important modification beyond epigenetics. However, systems-wide analyses of protein methylation function lag behind compared to other modifications. Recently, thermal stability analyses have been developed which provide a proxy of a protein functional status. Here, we show that diverse molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells (mESC) as a model system, we show that Prmt5 regulates numerous mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms. Our data show for instance that methylation of Prmt5 substrates promotes the formation of stress granules. Moreover, we reveal a novel non-canonical function of Ezh2 in mitotic chromosomes and the organization of the perichromosomal layer and identify Mki67 as a putative novel Ezh2 substrate involved in this process. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding the role of this modification in pluripotency.