Periodic Reporting for period 1 - RNAPII biogenesis (Regulating RNAPII levels: gaining insight into the translation, assembly and transport of RNAPII subunits.)
Periodo di rendicontazione: 2023-09-01 al 2025-09-30
RNA polymerase II (RNAPII), the key enzyme responsible for the transcription of all protein-coding genes and many non-coding genes in eukaryotes. RNAPII is a heteromeric complex, consisting of twelve subunits (RPB1-RPB12). Emerging evidence from our lab, as well as from others, has suggested that the cellular abundance of RPB1 is tightly regulated in cells, and that it can act as a regulatory node for transcription. Nevertheless, the mechanisms involved in sensing and regulating RPB1 levels, and in extension RNAPII abundance, remained poorly understood.
The objective of the project was therefore to gain more insight into how RNAPII levels are regulated in human cells. To this end, I had proposed three work packages that focused on different steps in the RNAPII biogenesis pathway, including synthesis of the RPB1 subunit synthesis, assembly of the entire RNAPII heteromeric complex, and its transport into the nucleus.
The objective of the project was therefore to gain more insight into how RNAPII levels are regulated in human cells. To this end, I had proposed three work packages that focused on different steps in the RNAPII biogenesis pathway, including synthesis of the RPB1 subunit synthesis, assembly of the entire RNAPII heteromeric complex, and its transport into the nucleus.
During the course of this action, we have achieved several important milestones. Specifically, we have identified a group of RNA-binding proteins that interact with the RPB1 mRNA and are likely involved in its post-transcriptional regulation. We are currently following up on these candidates with functional assays to gain further insight into their role in regulating RPB1 levels. In addition, we confirmed that a conserved stretch of nucleotides in the 3’UTR of RPB1 is important for the regulation of RPB1 mRNA, and have preliminary data suggesting that it might be important for the transcriptional activity of RNAPII. Moreover, we have uncovered that RNAPII subunits are assembled co-translationally in human cells, and are in the process of elucidating the precise mechanism by which this occurs.
We have generated several rich datasets that have provided insight into the proteins that are involved in the post-transcriptional regulation of RPB1, the main subunit of RNAPII. Moreover, we have generated cell lines, which have improved upon the current tools available to study the assembly of RNAPII in vivo. These tools have been invaluable in gaining insight into the biogenesis of RNAPII and have opened up further avenues for mechanistic research that are currently being explored. These findings not only provides a better understanding of transcription, but also open up new avenues for therapeutic strategies. For example, different modes of inhibition of RNAPII-mediated transcription are currently investigated as a potential therapy for cancer.