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Imaging dynamic interactions between spliceosomal proteins and pre-mRNA in living cells

Author:
Rino, José, Martin, Robert M., Carvalho, Teresa, Carmo-Fonseca, Maria
Source:
Methods 2014 v.65 pp. 359-366
ISSN:
1046-2023
Subject:
RNA, color, diffusivity, dissociation, fluorescence microscopy, fluorescence recovery after photobleaching, image analysis, mammals, proteins, spliceosomes
Abstract:
The ability to observe protein dynamics in living cells is critical for the mechanistic understanding of highly flexible biological processes such as pre-mRNA splicing by the spliceosome. Splicing relies on intricate RNA and protein networks that are repeatedly rearranged during spliceosome assembly. Here we describe a method based on fluorescence microscopy that has been used by our and other laboratories to study interaction of spliceosomal proteins with nascent pre-mRNA in living cells. The method involves co-expressing in mammalian cells the target pre-mRNA labeled with one color, and the spliceosomal protein tagged with another color. The diffusion coefficient of the protein as well as its association and dissociation rates with the pre-mRNA are estimated by fluorescence recovery after photobleaching (FRAP) or photoactivation.
Agid:
5537910