Oral Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

Human RNA Polymerase II is maintained in excess for robust transcriptional homeostasis (127463)

Alexander Gillis 1 2 3 , Scott Berry 1 2 3
  1. UNSW RNA Institute, Sydney
  2. EMBL Australia Node in Single Molecule Science, Sydney
  3. Molecular Medicine, University of New South Wales, Sydney

RNA polymerase II (Pol II) is the multi­protein complex responsible for transcribing all protein­coding messenger RNA (mRNA). Maintenance of the cellular concentration of mRNA is a key homeostatic parameter, and current evidence in yeast supports a limiting factor model in which the Pol II abundance of a cell dictates transcription level and therefore mRNA concentration. However, whether metazoan cells regulate Pol II abundance in order to control global transcriptional activity has not been tested.

 

We have recently demonstrated the critical role of a CRL3­ARMC5 ubiquitin ligase in degrading Pol II, regulating its cellular abundance during homeostasis. Using inducible ARMC5 degradation combined with imaging-­based assays of Pol II and of nascent transcription, we show that excess Pol II is not permitted into active elongation suggesting that, in contrast to yeast, human cells primarily control mRNA transcription downstream of Pol II abundance.

 

To further examine how mRNA production is globally controlled, we employ monoallelic degron tagging to induce partial depletion of Pol II. We tag each allele with independent fluorophores enabling their precise quantification at the single-­cell level. Intriguingly, Pol II transcriptional activity is extremely robust to this perturbation, being unchanged despite total Pol II levels being substantially reduced. This robustness mechanism has at least two dynamic phases. The first, on a time­scale of tens of minutes, is mediated through increased binding of Pol II onto chromatin from the nucleoplasmic pool, and the second, on a time­scale of several hours, is mediated through increased abundance of the non­degraded allele leading to compensatory restoration of Pol II levels back to almost baseline levels.

 

We employ live cell imaging of Pol II protein dynamics, including single-particle tracking, to demonstrate the level of Pol II stably bound to chromatin and elongating is maintained by controlled loading from the freely diffusing, inactive Pol II pool. By combining orthologous imaging approaches, including those with single molecule resolution, we examine the timescales of distinct Pol II states.

 

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.