RNA polymerase II (Pol II) is composed of 12 subunits that collaborate to synthesize mRNA within the nucleus. Pol II is widely recognized as a passive holoenzyme, with the molecular functions of its subunits largely ignored. Recent studies employing auxin-inducible degron (AID) and multi-omics techniques have revealed that the functional diversity of Pol II is achieved through the differential contributions of its subunits to various transcriptional and post-transcriptional processes. By regulating these processes in a coordinated manner through its subunits, Pol II can optimize its activity for diverse biological functions. Here, we review recent progress in understanding Pol II subunits and their dysregulation in diseases, Pol II heterogeneity, Pol II clusters and the regulatory roles of RNA polymerases.