visualizing-tracking-endogenous-mrnas-live-drosophila-melanogaster

Visualizing and Tracking Endogenous mRNAs in Live <em>Drosophila melanogaster</em> Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

CUNY School of Medicine

Graduate Center, City University of New York

City University of New York

Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF. 

Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber.

To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. We report for the first time the expression patterns during oogenesis for Drongo, the fruit fly homolog of AGFG1. We find that temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. Here we show a first link between the recycling endosome pathway and Drongo, and find that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.

The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11.

Molecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program () that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches.  takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an  mRNA-specific molecular beacon (osk1236), which is selected by  to target a more accessible region than a manually designed -specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting  mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

Article	Year
Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26324434	2015
The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA biology\| PubMed ID: 27654348	2016
: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs. RNA (New York, N.Y.)\| PubMed ID: 30573696	2019

Livia V. Bayer

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