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
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
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
