Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

Abstract

ISH: In situ hybridization DMD: Duchenne muscular dystrophy BMD: Becker muscular dystrophy NMD: Nonsense-mediated decay DAGC: Dystrophin-associated glycoprotein complex nNOS: neuronal nitric oxide synthase PPMO: PIP6a peptide-conjugated antisense morpholino oligonucleotide FITC: Fluorescein isothiocyanate Cy3/Cy5: Cyanine 3/5 SDH: Succinate dehydrogenase PTC: Premature termination codon AbstractDystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcripts at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We present a novel approach, instead using RNAscope probes targeted to 5' and 3' regions of the same transcript: labelling muscle dystrophin mRNA in this manner allows transcriptional dynamics to be deciphered in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs (the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggests this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

“…Used in combination, these three probes allow a high degree of isoform discrimination within a single sample: only dp427 will label with all three probes, thus presence of 5’ probe indicates full-length dystrophin expression. Given the ~16 hour transcription time, concerted expression of dp427 will result in many nascent transcripts as shown previously 42 : 5’ signal will be predominantly in the form of large nuclear foci, accompanied by progressively smaller foci of middle and 3’ probes ( Figure 1H ). Presence of middle and 3’ probes in the absence of 5’ probe ( Figure 1I ) indicates dp260 or dp140, with middle probe likely to form smaller nuclear foci (commensurate with the predicted ~9.5- and ~8-hour transcription times of dp260 and dp140, respectively, assuming consistent transcription at ~40 bases.sec -1 45 ).…”

“…Fresh-frozen canine skeletal muscle . Preparation of frozen muscle sections for RNAscope necessitates extended fixation times and an additional baking step as described previously 42 : slides were removed from -80°C freezer and placed immediately into cold (4°C) 10% neutral-buffered formalin, then incubated at 4°C for 1 hour. Slides were dehydrated in graded ethanol series (50%, 70%, 100% x2, 5 mins in each, room temperature) then air-dried and baked at 37°C for 1 hour.…”

“…Unless otherwise indicated, 20x objectives were used: this magnification was sufficient to resolve discrete spots corresponding to individual transcripts, while retaining adequate depth of focal plane to allow all elements of the tissue section to remain well-focussed. Where used (skeletal muscle samples only), image analysis was conducted using automated ImageJ macros as described previously 42 . All macros are in .ijm format, written for the Fiji distribution of ImageJ, and are available at the Figshare repository (see Underlying data 50 ).…”

“…We have modified this multiplex strategy yet further: even with target sequences 1000 bases in length, the 14 kb of the dp427 transcript permits multiple probes to bind, allowing dystrophin transcripts to be labelled with multiple fluorophores in a sequence-specific manner. We have previously employed this multiplex strategy in healthy and dystrophic skeletal muscle 42 using probes to the 5’ and 3’ regions of dp427, regions that, given the length of the transcript and the 16-hour transcription time, are separated both spatially and temporally (5’ sequence is transcribed several hours before 3’ sequence 3 , 43 ). In healthy mouse muscle ( Figure 1F ), punctate sarcoplasmic fluorescent foci of both 5’ and 3’ probes are found in close proximity, indicating dual-labelling of mature, exported dp427m transcripts, while the 5’ probe also produces broad and intensely fluorescent foci within nuclei, indicating the presence of many nascent dp427 mRNAs (20-40 per myonucleus 42 ) arrayed along the dystrophin locus (correspondingly, small nuclear 3’ foci are observed infrequently).…”

“…We have previously employed this multiplex strategy in healthy and dystrophic skeletal muscle 42 using probes to the 5’ and 3’ regions of dp427, regions that, given the length of the transcript and the 16-hour transcription time, are separated both spatially and temporally (5’ sequence is transcribed several hours before 3’ sequence 3 , 43 ). In healthy mouse muscle ( Figure 1F ), punctate sarcoplasmic fluorescent foci of both 5’ and 3’ probes are found in close proximity, indicating dual-labelling of mature, exported dp427m transcripts, while the 5’ probe also produces broad and intensely fluorescent foci within nuclei, indicating the presence of many nascent dp427 mRNAs (20-40 per myonucleus 42 ) arrayed along the dystrophin locus (correspondingly, small nuclear 3’ foci are observed infrequently). In dystrophic mdx muscle ( Figure 1G ) sarcoplasmic foci (5’ and 3’) are dramatically reduced as expected: mature dp427 transcripts are rapidly degraded by nonsense-mediated decay (NMD), a process that occurs after nuclear export 44 .…”