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RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3′ end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3′ end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells. [Display omitted] • Disassociated RPB3 (dRPB3) exists in various cells • dRPB3 preferentially controls 3′ end processing of ribosomal subunit genes • The RPB3 N terminus directly interacts with CBC and is required for 3′ end processing • LEC is associated with the specificity of the dRPB3-mediated CBC-PCF11 axis Li et al. show that disassociated RPB3 imparts the preferential regulation of 3′ end processing for ribosomal subunit genes. This indicates that RNA Pol II subunits cause RNA Pol II to transform from a mono-functional enzyme to a multi-functional enzyme, increasing enzyme efficiency and conferring specificity regulations. [ABSTRACT FROM AUTHOR]