Multiplex in situhybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics.

John C W Hildyard,Dominic J Wells,Faye Rawson,Richard J Piercy

doi:10.1371/journal.pone.0239467

Abstract

Dystrophin 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 transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with 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 suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Highlights

The dystrophin gene is the largest in the genome: at approximately 2.4Mbp in length this single gene accounts for almost 0.1% of human haploid DNA and occupies fully 1.5% of the X chromosome where it resides
RNAscope in-situ labelling allows resolution of individual mRNA molecules, when used with low-abundance transcripts [24], and the expected staining pattern is that of small, punctate foci with an apparent diameter of 1–1.5μm distributed throughout the cell interior
Single transcript multiplex in-situ hybridization (ISH) (5’ and 3’) reveals dystrophin transcriptional dynamics, showing that even in healthy muscle most dp427 is nascent, and that mature transcripts are remarkably short-lived

Summary

Introduction

The dystrophin gene is the largest in the genome: at approximately 2.4Mbp in length this single gene accounts for almost 0.1% of human haploid DNA and occupies fully 1.5% of the X chromosome where it resides. Transcription of a single full-length 79-exon dystrophin mRNA is estimated to take 16 hours, with translation giving rise to dp427 dystrophin (a protein product of 427kDa). This lengthy gene displays unconventional transcriptional behaviour: the dp427 transcript can arise from three separate promoters controlling cortex, muscle and Purkinje cell expression, producing dp427c, dp427m and dp427p respectively [1, 2]. Each promoter confers a unique first exon but the other 78 are shared, resulting in dp427 proteins

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Conclusion