Journal
NUCLEIC ACIDS RESEARCH
Volume 41, Issue 1, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gks837
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Funding
- National Institutes of Health (NIH) [CA147922, GM094198]
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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 beta-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.
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