Abstract:
:mRNA expression involves transcription initiation, elongation and degradation. In cells, these dynamic processes are highly regulated. However, experimental characterization of the dynamic processes in vivo is difficult due to the paucity of methods capable of direct measurements. We present a highly sensitive and versatile method enabling direct characterization of the dynamic processes. Our method is based on single-molecule fluorescence in situ hybridization (smFISH) and quantitative analyses of hybridization signals. We hybridized multiple probes labelled with spectrally distinct fluorophores to multiple sub-regions of single mRNAs, and visualized the kinetics of synthesis and degradation of the sub-regions. Quantitative analyses of the data lead to absolute quantification of the lag time of mRNA induction (the time it takes for external signals to activate transcription initiation), transcription initiation rate, transcription elongation speed (i.e. mRNA chain-growth speed), the rate of premature termination of transcripts and degradation rates. Applying our method to three different biological problems, we demonstrated how our method may be applicable to reveal dynamics of mRNA expression that was difficult to study previously. We expect such absolute quantification can greatly facilitate understanding of gene expression and its regulation working at the levels of transcriptional initiation, elongation and degradation.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Iyer S,Park BR,Kim Mdoi
10.1093/nar/gkw596subject
Has Abstractpub_date
2016-10-14 00:00:00pages
e142issue
18eissn
0305-1048issn
1362-4962pii
gkw596journal_volume
44pub_type
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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