Identification of the molecular mechanism of transcription regulation which monitors the quantity and quality of Pol II transcription complexes on genes
In eukaryotic cells, transcription of the coding genome is conducted by RNA Polymerase II (Pol II), a multi-subunit enzyme which uses a DNA template to synthesise complementary copies of RNA. This transcription is the first step in the expression of all protein-coding genes, so regulation of this process is key to virtually all cellular activities. To ensure their continued survival, cells must therefore strictly regulate Pol II activity and abundance in advance of transcription beginning, allowing them to eliminate proteins that become dysfunctional or excessive. Ana Tufegdžić Vidaković’s group, in the LMB’s PNAC Division, have worked with Scott Berry’s group at the University of New South Wales, Australia, to identify the quality control mechanism which human cells use to target defective Pol II molecules for degradation.
Specifically, the group found that upon defects in activity, Pol II is targeted for degradation by the E3 ubiquitin ligase CRL3 with the substrate recognition factor ARMC5. Together, they add a chain of ubiquitin molecules onto a subunit of Pol II. This results in the removal of defective Pol II. Moreover, the group found that degradation of Pol II molecules at the onset of transcription was very common. This suggests that transcription-initiating Pol II is prone to defects and thus the quality checkpoint at this stage is vital.
This new transcriptional mechanism was elucidated by Roberta Cacioppo, postdoc in Ana’s group, Alexander Gillis, postdoc in Scott’s group, Ivan Shlamovitz, a Ph.D. student in Ana’s group and Andrew Zeller, a Ph.D. student in Julian Sale’s group (also in the LMB’s PNAC Division).
First, Roberta induced defects in Pol II activity and analysed the ubiquitylation profile of Pol II, revealing the key role of ARMC5 in this process. Concurrently, Alexander used immunofluorescence and live-cell microscopy to confirm the role of ARMC5 in controlling Pol II half-life and stability. Complementary assays in Scott’s and Ana’s groups then revealed an intriguing Pol II behaviour when ARMC5 was lost. Alexander used live cell microscopy, Roberta used biochemistry and Ivan and Andrew used genomics, all to demonstrate that in the absence of ARMC5, excess Pol II molecules accumulated both in the nuclear solution and in the region of genes where transcription begins – but surprisingly, this didn’t result in increased transcription of genes. Instead, the excess Pol II molecules were constrained in a region in the beginning of the gene. This suggested the presence of a secondary checkpoint, working to limit the quantity of Pol II molecules that is allowed to proceed into the gene and complete transcription.
To identify this compensatory checkpoint, Roberta used a synthetic lethality mini-screen, and found the Integrator phosphatase module subunit 8 (INTS8) prevented the excess Pol II molecules from moving beyond transcription start sites. This therefore compensated for the loss of ARMC5. The group then analysed Pol II’s ability to transcribe in the absence of both ARMC5 and INTS8 – this resulted in uncontrolled release of Pol II into genes, resulting in severe impairment of transcription and cellular growth. Furthermore, these escaped Pol II molecules were incompetent and failed to reach gene ends, likely because they had not been surveyed by the ARMC5-mediated quality control mechanism at the beginning of transcription.
This work reveals a new mechanism of transcription regulation via control of quality and quantity of Pol II molecules. Determining the key role of ARMC5 may help in understanding a rare disease affecting those with germline mutations in the ARMC5 gene, primary bilateral macronodular adrenal hyperplasia. The work also raises interesting questions for future study, for example, how do ARMC5 and INTS8 “know” which Pol II molecules to target? Both factors have been previously defined as tumour suppressor genes, so further research into their mechanism of action may inform future therapeutic approaches.
This work was funded by UKRI MRC, the Australian Research Council, the Darwin Trust of Edinburgh, Boehringer Ingelheim Fonds, and Cancer Research UK.
Further references
CRL3ARMC5 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of Pol II transcription. Cacioppo, R., Gillis, A., Shlamovitz, I., Zeller, A., Castiblanco, D., Crisp, A., Haworth, B., Arabiotorre, A., Abyaneh, P., Bao, Y., Sale, J.E., Berry, S., Tufegdžić Vidaković, A. Molecular Cell
Ana’s group page
Scott Berry – University of New South Wales