RNA polymerase II (Pol II) is the multiprotein complex responsible for transcribing all protein-coding messenger RNA (mRNA). Maintenance of the cellular concentration of mRNA is a key homeostatic parameter, however the extent to which metazoan cells control global mRNA transcription by regulating Pol II has not been established.
We have recently demonstrated the critical role of a CRL3ARMC5 ubiquitin ligase in regulating cellular abundance of Pol II. Using inducible ARMC5 degradation combined with assays of nascent transcription and of Pol II protein dynamics, including single particle tracking (SPT), we show that excess Pol II is not permitted into active elongation but rather is held in the inactive, freely diffusing pool.
To further examine the control of mRNA production, we induce partial depletion of Pol II using monoallelic degron tagging, labelling each allele with independent fluorophores enabling precise single-cell quantification. Intriguingly, Pol II transcriptional activity measured both via transient transcriptome sequencing (TT-seq), and via imaging methods, is extremely robust to this perturbation, being unchanged despite total Pol II levels being substantially reduced.
The mechanism of this robustness has at least two separate dynamic phases. By integrating information from Pol II chromatin-immunoprecipitation followed by sequencing (ChIP-seq) and live cell assays of Pol II binding, we establish a rapid initial phase of adaptation, where a reduced pool of Pol II is able to maintain total transcriptional output, is mediated by increased binding of Pol II and release to elongation. A second, slower adaptation mechanism is mediated through increased abundance of the nondegraded allele, leading to compensatory restoration of Pol II levels back to almost baseline levels.
These data demonstrate that human cells have multiple layers of control over ongoing mRNA transcription by Pol II, by adjusting both abundance and activity to maintain homeostasis.