Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution

Abstract

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.

Parallel monitoring of mRNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

“…While the method appears to work relatively well in large, cultured mammalian cells, its limits become in particular apparent when working with smaller cells such as yeast and many protozoans, that have a cell wall or dense cytoskeleton underneath the plasma membrane. In trypanosomes for example, there is a huge variance in fluorescence intensities between mRNA molecules within the same cell 2,3 and the same is observed on recent smFISH images from Plasmodium 4 . Almost no yeast publications exist that use branched DNA technology.…”

“…In branched DNA technology, the RNA molecule is detected by a set of up to 20 probe pairs, each consisting of two adjacent oligos that further hybridise in several steps with other oligos to generate a large branched structure of DNA finally linked to fluorophores 1 . Branched DNA technology has two major advantages in comparison to standard smFISH: (1) background fluorescence is massively reduced, as each signal depends on hybridisation of two oligos next to each other (binding of just one oligo will not result in a signal) and (2) the amplification of the signal results in labelling with up to 400 fluorophores per probe pair and thus reduces the minimal size of the target sequence to about 50 nucleotides (recognition site of one probe pair). This allows probing for very small RNAs or distinguishing RNAs with minor sequence differences, for example alternative splicing products.…”

mRNA detection in budding yeast with single fluorophores

“…Our sFISH method not only offers a time- and cost-efficient mRNA quantification tool, but also opens up new opportunities for transcript research at the single cell level. Owing to its much smaller target size compared to mFISH, sFISH can be used to detect RNA shorter than 100 nt, profile mRNA isoforms ( 38 , 59 , 60 ), detect subtle changes in mRNA sequence over a 30-nt window as a result of alternative splicing ( 61 – 63 ) and to measure transcriptional ( 64 ) and degradational intermediates ( 65 ) with improved resolution. FRET-FISH, which we demonstrated here, can also be used to detect circular RNA ( 66 ) in situ without an amplification step.…”

Trypanosomes can initiate nuclear export co-transcriptionally

“…smRNA FISH was done as previously described (39). The sequences used to design the Affymetrix FISH probes are in Supplementary Table S2.…”