Periodic Reporting for period 1 - PhosphoMLO (Regulation of membraneless organelles through phosphatase-DYRK kinase feedback)
Reporting period: 2022-09-01 to 2024-08-31
Biological molecules like proteins and RNAs interact within cells to perform specific functions. Some of these inter-molecular protein-RNA interactions lead to the formation of compartments that are separated from the rest of the cell. These compartments broadly function to segregate components from the rest of the cell or concentrate reaction machinery to promote specific processes. Recent research continues to reveal the presence of new compartments driven by loose interactions among disordered regions of proteins, as well as elucidate the functions of known compartments. For example, although nuclear speckles are conserved across cell types throughout the animal kingdom and dysregulation of their components occurs in human diseases including cancer and neurodegenerative disease, their exact functions are still unclear. By uncovering the modulators of nuclear speckles and other condensates in human cells, we can target their properties and observe cellular changes to gain insight into their functions. Understanding the normal regulation and function of cellular processes is crucial to developing methods to reverse disease-mediated changes. The overall objectives of the project were to 1) identify enzymatic regulators of condensates, 2) characterize the interactions between enzymatic regulators and their target condensates, and 3) define the relationships among opposing regulators.
For this project, we ran a microscopy-based screen to uncover phosphatases that regulate various condensates. We found that the PP1 phosphatases, a family of broadly acting serine-threonine phosphatases, maintain a cohesive state in the prominent nuclear condensate nuclear speckles and target similar proteins to the kinase DYRK3. Pushing nuclear speckles to a more dephosphorylated state decreased the rate at which speckle proteins components were lost and gained from their surroundings. At the same time, we found that speckles retain more mRNA, essentially sequestering mRNA within the nucleus where it can’t be translated into proteins. To determine whether nuclear speckle-mediated regulation of mRNA retention affects particular genes, we ran a proximity labelling experiment while modulating the phosphorylation of nuclear speckles. Verifying the results by imaging and quantifying the localization of specific transcripts, we found that although certain functional classes of mRNAs were more enriched within speckles at baseline, speckle dephosphorylation resulted in broad retention of mRNAs across the transcriptome. After identifying the phosphatase regulators of speckles, we then further investigated conditions that could affect phosphatase activity and the possible contributions of regulatory subunits using immunofluorescence and live-cell imaging. We found that dephosphorylation increases under heat shock and oxidative stress and decreases under hypoxic conditions, which was reflected in expected changes in the nuclear retention of mRNAs, and may contribute to broad changes in translation under these conditions. Many human diseases involve either increased oxidative stress or hypoxia, therefore understanding changes in mRNA regulation under these conditions may have future clinical implications.
Additional work also identified PP1 phosphatases as regulators of a condensate in the pathway that cells to secrete molecules. Decreasing phosphorylation of this condensate impeded functional secretion while decreasing the rate at which speckle proteins components were lost and gained from their surroundings, similar to the effects we observed on nuclear speckles.
The results of the project have so far been published as a preprint, and also contributed to a peer-reviewed publication for dissemination to the scientific community.
Additional work also identified PP1 phosphatases as regulators of a condensate in the pathway that cells to secrete molecules. Decreasing phosphorylation of this condensate impeded functional secretion while decreasing the rate at which speckle proteins components were lost and gained from their surroundings, similar to the effects we observed on nuclear speckles.
The results of the project have so far been published as a preprint, and also contributed to a peer-reviewed publication for dissemination to the scientific community.
The overall objectives for the project were to identify the molecular regulators of biomolecular condensates, then characterize their effects and interactions. Based on our data, we identify PP1 phosphatases and the kinase DYRK3 as co-regulators of two condensates, nuclear speckles and a newly described condensate in the secretory pathway of the cell. We show that proteins within nuclear speckles lose modifications under cellular stress conditions, enabling slower exchange of component proteins with the surrounding fluid and higher retention of mRNAs. When proteins involved in the formation of secretory condensates similarly lose modifications, these condensates also enter a more gel-like state and cellular secretion fails. The project provides a basis to further investigate how the material properties of various condensates affect their functions under diverse cellular conditions. In particular, these results raise additional questions on whether the perturbations to nuclear speckle protein interactions observed in the context of cancer or neurodegenerative disease affect mRNA retention in similar ways to those uncovered here, and whether the molecular regulators PP1 and its regulatory subunits or DYRK3 could be targeted for the development of new therapeutics.