Real-time imaging of gene expression in living cells
Rino J., Martins M., Carvalho C., Braga J., Carvalho T. and Carmo-Fonseca, M.
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal
During the last decade, technical advances in the field of live-cell imaging provided biophysicists with revolutionary tools to visualize and quantify the kinetics and interactions of molecules inside individual living cells. We are interested in the biological process of mRNA biogenesis, particularly in the splicing reaction, which consists in the removal of non-coding sequences (introns) from the pre-mRNA transcript. Splicing is performed by the spliceosome, a very dynamic,multicomponent macromolecular machine. Ample evidence from a series of biochemical experiments demonstrates that splicing is tightly coupled to transcription, with introns being excised while the nascent transcript is still associated with the gene. However, the nature of this coupling and the kineticcrosstalk between the two processes still remain largely unknown.With the aim of identifying the rate limiting steps in co-transcriptional mRNA biogenesis and in particular the influence of splicing, we have established a live cell microscopic assay that allows for real-time simultaneous imaging of transcription and splicing kinetics. Our system consists ofinducible cell lines which containa stablyintegrated tandem array of a given gene of interest. This gene codes for mRNAs whichharbor binding sites for a red fluorescently tagged protein.Transcription, on the other hand, is carried out in these cells exclusively by a RNA Polymerase II tagged with the green fluorescent protein (GFP).Using FRAP (Fluorescence Recovery After Photobleaching), we are able to simultaneously measure the dynamics of GFP-tagged Pol II and red fluorescently-labeled RNA transcripts. Our results show that the turnover of RNA transcripts at the site of transcriptionis faster than that of RNA Polymerase II, suggesting a more stable association of Pol II with the gene template. Most important, in the presence of a spliceosome assembly inhibitory drug, the dynamics of RNA Pol II is significantly faster than in the absence of the drug. These results argue that spliceosome assembly slows down the kinetics of transcription by RNA pol II.
Short biographical note
José Rino graduated in Technological Physics Engineering at Instituto Superior Técnico in 1999. He received a Ph.D. in Biophysics from the University of Lisbon (2007) after a doctoral thesis at the Cell Biology Unit of Instituto de Medicina Molecular (IMM), where his work has been focused on the development of microscopy tools to study the interactions and dynamics of proteins and RNA inside living cells. He is currently Head of the BioImaging Unit at the IMM.