Abstract
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.
| Original language | English |
|---|---|
| Article number | e1008459 |
| Journal | PLoS Genetics |
| Volume | 15 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 2019 |
Bibliographical note
Publisher Copyright:© 2019 Hochberg-Laufer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding
This work was supported by the German-Israeli Foundation (929/06) and the European Research Council (GENEXP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Eva Böhnlein and Karla Neugebauer (Yale University) for generating the SF-GFP BAC cell lines. We thank Jennifer I.C Benichou (BIU) for the assistance with the statistical analysis and Noa Kinor for assistance with experiments and arrangement of the figures.
| Funders | Funder number |
|---|---|
| GENEXP | |
| Yale University | |
| European Commission | |
| German-Israeli Foundation for Scientific Research and Development | 929/06 |
