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Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation.

ABSTRACT: The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA-protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with ?-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.


PROVIDER: S-EPMC3592441 | BioStudies | 2013-01-01T00:00:00Z

REPOSITORIES: biostudies

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