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

The molecular beacon technology is an established approach for visualizing native mRNAs in living cells. These probes need to efficiently hybridize to accessible RNA regions in order to spatially and temporally resolve the dynamic steps of the RNA life cycle. A refined method using two computer algorithms, mfold and RNAstructure, is described for choosing shorter, more abundant target regions for molecular beacon binding. The probes are redesigned as small hairpins and are synthesized from 2'-O-methyl RNA/LNA chimeric nucleic acids. These tiny molecular beacons are stable in the cellular environment and have a high affinity for binding to target RNAs. The user-friendly synthesis protocol and ability to couple to a variety of fluorophores make tiny molecular beacons the optimal technology to detect less abundant, highly structured RNAs, as well as small RNAs, such as microRNAs. As an example, tiny chimeric molecular beacons were designed to target regions of oskar mRNA, microinjected into living Drosophila melanogaster oocytes and imaged via spinning disc confocal microscopy.

Tiny molecular beacons for in vivo mRNA detection.

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
Tiny molecular beacons for in vivo mRNA detection. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21431739	2011
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

Diana P. Bratu

Biography

JoVE Journal Publications

Other Publications