Availability of splicing factors in the nucleoplasm can regulate the release of mRNA from the gene after transcription

Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the 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. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing. Author summary Genetic information is contained in the cell nucleus and encodes proteins. However, protein production takes place in the cytoplasm, and so a molecule is needed to connect between the nucleus and cytoplasm. This messenger molecule is called messenger RNA (mRNA). It is produced and copied from the DNA, and after some processing will travel to the cytoplasm to encode proteins. This study focuses on the timing of mRNA release from the gene after it is copied from the DNA. Processing of mRNA includes the removal of some of its pieces and the stitching back of the remaining parts. This is called splicing. We found that mRNAs undergoing many splicing events are retained on the gene until splicing has completed, and examined what is the cause for this delay. We found that the factors performing the splicing might be limiting the process if their levels are not high enough at the gene locus. By increasing splicing factor levels in the nucleus we show that their abundance increases the rate at which mRNA is released. This means that the cell can regulate gene expression rates by limiting the availability of splicing factors that are free to take part in the processing of mRNA.