A protocol for in vivo RNA labeling and visualization in tobacco pollen tubes
Year: 2024
Type: article
Abstract: Here, we present a protocol for labeling and live visualization of RNA-protein complexes in the form of ribonucleoprotein particles (RNPs) in tobacco pollen tubes. We describe steps for constructing RNA-pp7/MS2 tag and biolistic gene-gun-mediated pollen transformation. We then provide detailed procedures for RNA labeling using PP7 aptamer nascent RNA tagging and a fluorescently labeled Pseudomonas aeruginosa PP7 bacteriophage coat protein (PCP) reporter that binds to PP7 RNA stem loops. This protocol is adaptable to other cell types by employing tissue-specific promoters. • Instructions for generation of fluorescence in vivo PP7 aptamer RNA labeling • Steps for visualizing RNP complexes in transformed tobacco pollen tubes • Tips for checking the specificity of aptamer-reporter RNPs in pollen tubes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for labeling and live visualization of RNA-protein complexes in the form of ribonucleoprotein particles (RNPs) in tobacco pollen tubes. We describe steps for constructing RNA-pp7/MS2 tag and biolistic gene-gun-mediated pollen transformation. We then provide detailed procedures for RNA labeling using PP7 aptamer nascent RNA tagging and a fluorescently labeled Pseudomonas aeruginosa PP7 bacteriophage coat protein (PCP) reporter that binds to PP7 RNA stem loops. This protocol is adaptable to other cell types by employing tissue-specific promoters.
Source: STAR Protocols
Authors: Vinod Kumar, Said Hafidh
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4404404907