Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers.

Weibing Yang,Nathanaël Prunet,Christoph Schuster,Qingkun Dong,Raymond Wightman,Elliot M Meyerowitz,Benoit Landrein

doi:10.1104/pp.19.00980

Abstract

In addition to transcriptional regulation, gene expression is further modulated through mRNA spatiotemporal distribution, by RNA movement between cells, and by RNA localization within cells. Here, we have adapted RNA fluorescence in situ hybridization (FISH) to explore RNA localization in Arabidopsis (Arabidopsis thaliana). We show that RNA FISH on sectioned material can be applied to investigate the tissue and subcellular localization of meristem and flower development genes, cell cycle transcripts, and plant long noncoding RNAs. We also developed double RNA FISH to dissect the coexpression of different mRNAs at the shoot apex and nuclear-cytoplasmic separation of cell cycle gene transcripts in dividing cells. By coupling RNA FISH with fluorescence immunocytochemistry, we further demonstrate that a gene's mRNA and protein may be simultaneously detected, for example revealing uniform distribution of PIN-FORMED1 (PIN1) mRNA and polar localization of PIN1 protein in the same cells. Therefore, our method enables the visualization of gene expression at both transcriptional and translational levels with subcellular spatial resolution, opening up the possibility of systematically tracking the dynamics of RNA molecules and their cognate proteins in plant cells.

Highlights

With the rapid development of sequencing technologies and continuing reduction in sequencing cost, a growing number of plant genome sequences are being released, and de novo assembly of model and nonmodel plant genomes is becoming feasible for individual research groups (Jiao and Schneeberger, 2017; Michael et al, 2018)
We have established RNA fluorescence in situ hybridization (FISH) in the Arabidopsis shoot apex based on the tyramide signal amplification (TSA) system applied to sectioned tissue (Yang et al, 2017)
We proposed that a feasible strategy would meet the following criteria: (1) specificity: fluorescence signals need to reliably reflect the localization of target RNA molecules; (2) sensitivity: variation in gene expression levels requires a flexible detection method that is suitable for both high- and low-abundance transcripts; and (3) scalability: such a method should be achievable for examining and comparing multiple transcripts in a cost- and time-effective manner

Summary

Introduction

With the rapid development of sequencing technologies and continuing reduction in sequencing cost, a growing number of plant genome sequences are being released, and de novo assembly of model and nonmodel plant genomes is becoming feasible for individual research groups (Jiao and Schneeberger, 2017; Michael et al, 2018). To determine RNA localization at the cellular level, fluorescence in situ hybridization (FISH) was developed and has become a vital tool in analyzing animal and yeast gene expression (Levsky and Singer, 2003). We provide step-by-step protocols showing how mRNAs of different genes can be concurrently analyzed through double RNA FISH, and we combine RNA FISH with immunofluorescence to visualize both mRNA and the corresponding protein within the same sample. We demonstrate these techniques by mapping the mRNA distribution and coexpression of flower development genes, investigating a novel family of nuclear mRNAs, and exploring the correlation of PIN-FORMED1 (PIN1) transcription and protein polarization in thick shoot tissues

Methods

Results

Conclusion