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2017 ; 45
(7
): e49
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Simultaneous detection of mRNA transcription and decay intermediates by dual
colour single mRNA FISH on subcellular resolution
#MMPMID27940558
Kramer S
Nucleic Acids Res
2017[Apr]; 45
(7
): e49
PMID27940558
show ga
The detection of mRNAs undergoing transcription or decay is challenging, because
both processes are fast. However, the relative proportion of an mRNA in synthesis
or decay increases with mRNA size and decreases with mRNA half-life. Based on
this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous
mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5? and 3?
ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH
probes, respectively. In the resulting fluorescence images, yellow spots
represent intact mRNAs; red spots are mRNAs in transcription or 3?-5? decay, and
green spots are mRNAs in 5?-3? degradation. Most red spots were nuclear and
insensitive to transcriptional inhibition and thus likely transcription
intermediates. Most green spots were cytoplasmic, confirming that the majority of
cytoplasmic decay in trypanosomes is 5?-3?. The system showed the expected
changes at inhibition of transcription or translation and RNAi depletion of the
trypanosome homologue to the 5?-3? exoribonuclease Xrn1. The method allows to
monitor changes in mRNA metabolism both on cellular and on population/tissue wide
levels, but also to study the subcellular localization of mRNA transcription and
decay pathways. I show that the system is applicable to mammalian cells.