The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how t...

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Bibliographic Details
Published in:The Journal of cell biology 2010-10, Vol.191 (1), p.75-86
Main Authors: Huranová, Martina, Ivani, Ivan, Benda, Aleš, Poser, Ina, Brody, Yehuda, Hof, Martin, Shav-Tal, Yaron, Neugebauer, Karla M., Staněk, David
Format: Article
Language:English
Subjects:
Catalysis
Cell Line
Cell lines
Cell nucleus
Cell Nucleus - metabolism
Cells
Exons
Fluorescence Recovery After Photobleaching
HeLa Cells
Humans
Introns
Kinetics
Proteins
Reaction kinetics
Ribonucleic acid
Ribonucleoproteins, Small Nuclear - metabolism
Ribonucleoproteins, Small Nuclear - physiology
RNA
RNA Precursors - metabolism
RNA Splicing - physiology
RNA, Messenger - metabolism
Small nuclear ribonucleoproteins
Small nuclear RNA
Spectrometry, Fluorescence
Spliceosomes
Spliceosomes - metabolism
Splicing
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Summary:Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.201004030