Nuclear condensates and gene expression regulation - probing the connection using live-cell imaging approaches

Session

Live and Functional Imaging Technologies Part 1

Authors

Yaron Shav-Tal (1)

Affiliations

1. The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan 52900, Israel

Abstract text

Nuclear speckles are membrane-less nuclear bodies that harbor RNA-binding proteins, particularly an abundance of splicing factors. The function of these nuclear condensates is unclear. Studies in living cells with fluorescently tagged splicing factors have led to a proposed model in which splicing factors can shuttle between nuclear speckles and transcribing genes, to participate in intron removal from pre-mRNAs. After completion of the splicing reaction, splicing factors can return to the nuclear speckles to be re-phosphorylated for the next cycle. To test the influence of nuclear speckles on transcription and splicing kinetics, we used a detectable, transcriptionally active gene in living cells. Using photobleaching measurements in living cells we could examine a) splicing factor recruitment dynamics at the active gene and also b) measure the rate at which the transcribed mRNA was released from the gene following transcription and splicing. We previously found that an mRNA undergoing many splicing events was retained at this gene until the completion of mRNA processing, and this delay in release from the gene was splicing dependent. To determine whether the reason for this retention was splicing factor availability, we disassembled nuclear speckles by overexpression of the Clk1 kinase, and measured splicing factor dynamics in the nucleus. Disassembly of nuclear speckles increased the diffusing fraction of splicing factors in the nucleoplasm, and reduced their residence times on the active gene. Importantly, the mRNA that was previously retained on the gene until completion of processing was now rapidly released. In contrast, a mutant inactive form of Clk1 and other treatments tested, did not affect the dynamics of mRNA release from the gene. Rather, faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. In conclusion, use of live-cell imaging and quantitative analysis of splicing factor dynamics in the cell nucleus leads us to suggest that nuclear speckles can buffer the availability of splicing factors in the nucleoplasm, and as membrane-less structures can rapidly respond and regulate the kinetics of mRNA release from the gene after processing.