Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation.
Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells.
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3, Canada; College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, PR China.
Xiaofei
X
Cheng
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3, Canada.
Ping
P
Deng
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3, Canada.
Hongguang
H
Cui
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3, Canada. Electronic address: aiming.wang@agr.gc.ca.
Aiming
A
Wang
D058948:Cell Tracking / Q000379:methods; D042541:Intracellular Space / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D011485:Protein Binding; D021381:Protein Transport; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins
BiFC; D-bodies; Double-stranded RNA; Fluorescent protein; Live-cell imaging; Positive-sense RNA virus; RDR6; RNA binding protein; SGS3; Viral replication complex