Abstract
Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA–protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA–protein interactions such as that between influenza viral protein NS1 and the 5’ UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA–protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA–protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.
Highlights
Intracellular monitoring of RNA–protein interactions is of great importance for understanding the posttranscriptional processes of gene expression, as well as the virus–host interactions
An mRNA of interest is tagged with an ms2 cassette while the MS2 coat protein is fused to a split fragment of a yellow fluorescent protein (YFP) variant (Venus), while the complementary portion of Venus is fused to an RNA-binding protein of interest
No red fluorescent protein (RFP) signal was detected for negative control co-transfections that did not include influenza viral mRNAs. These results indicated that the Aly and UAP56 adapter proteins were able to bind influenza viral mRNAs
Summary
Intracellular monitoring of RNA–protein interactions is of great importance for understanding the posttranscriptional processes of gene expression, as well as the virus–host interactions. TriFC was first built by Rackham and Brown based on the protein complementation technique which originally developed to study protein-protein interactions in living cells [5]. Using this approach, an mRNA of interest is tagged with an ms cassette while the MS2 coat protein is fused to a split fragment of a yellow fluorescent protein (YFP) variant (Venus), while the complementary portion of Venus is fused to an RNA-binding protein of interest. If the RNA-binding protein interacts with the RNA sequence of interest, the two separate Venus fragments unite, emitting a YFP signal
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