Methods for visualizing RNA processing and transport pathways in living cells

Abstract

Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs. Also, the picture emerges that RNA molecules travel through the nucleus at much faster speed, equaling that of free diffusion, than previously anticipated. Still, a major challenge proves to be the development of a microscopic detection technique that allows specific, in vivo, detection of low levels of RNA species by fluorescence in situ hybridization, without interfering fluorescent background signals derived from non-hybridized probe sequences and autofluorescent cell components. By applying photoactivatable caged fluorochrome-, molecular beacon-, or fluorescence resonance energy transfer (FRET)-based detection methods an important step in the future of living cell analysis has already been made.

Enod40, a Short Open Reading Frame–Containing mRNA, Induces Cytoplasmic Localization of a Nuclear RNA Binding Protein in Medicago truncatula

“…These approaches include microinjection of fluorescent RNA, in vivo hybridization of fluorescent oligonucleotides, and expression of fluorescent RBPs (Dirks et al, 2001;Pederson, 2001). Indirect labeling of mRNAs with GFP has been successfully applied in yeast cells and rat neurons (Bertrand et al, 1998;Rook et al, 2000).…”

Real-time Detection of Gene Expression in Cancer Cells Using Molecular Beacon Imaging: New Strategies for Cancer Research

“…Although previous studies showed the feasibility of detecting mRNAs and monitoring the transportation of RNAs in cells, the procedure for delivery of the molecular beacons through microinjection or by liposome delivery has made it difficult to apply this technology into broad research areas or into a routine clinical procedure (9)(10)(11)(12). A recent study showed that it is feasible to transfect a molecular beacon into living cells to detect doxorubicin-induced activation of p21 gene expression (13).…”

“…The feasibility of detecting intracellular mRNA has been examined in several laboratories (9)(10)(11)(12)(13). It has been shown that molecular beacons were able to visualize mRNA molecules in several human and animal cell lines after introducing into cells through microinjection or liposome delivery (9)(10)(11)14). It has also been shown that the detection limit of preformed molecular beacon/ h-actin mRNA duplexes microinjected into the cells is 10 mRNA molecules, suggesting that molecular beacon technology is a very sensitive method for detecting mRNAs in cells (9).…”

P53-dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy

“…This backbone has been reported to increase the half-life of beacons to a period of 2 days. 19 The final p21-beacon design included the hybridization sequence in the loop structure and had a melting temperature of 53.3˚C (Fig 1C).…”