RNA-IP

(Lo Wu FEBS J 2019 PMID: 31034708) Assay for interaction between the 5'- and 3'-ends of the genome bridged by the N protein

The assay was based on the methods of Huang et al. [36], with some modifications. DNA templates pBM25A and psBM25A were used for in vitro transcription with T7 poly- merase (Promega) in the presence of a biotin-UTP labeling NTP mixture (Roche Applied Science), as recommended by the manufacturer, to synthesize biotinylated RNA with 50- terminal 120 nt of genome or N subgenome, respectively. After purification, the biotinylated RNA was incubated with recombinant N protein and 32P-labeled RNA containing 30- terminal 55 nt plus the poly(A) tail in a reaction buffer con- taining 5 U of RNasin (Promega), 25 mM KCl, 5 mMHEPES (pH 7.6), 2 mM MgCl2, 0.1 mM EDTA, 0.2% glyc- erol, and 2 mM DTT. After incubation at room temperature for 30 min, a streptavidin suspension (MagQu, Taipei, Tai- wan) was added to the mixture and incubated for 30 min at room temperature followed by three washes with binding buffer. RNAs bound to streptavidin agarose beads were eluted by a buffer [7 M urea, 350 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS] followed by phenol- chloroform extraction and ethanol precipitation. The samples were resolved on a 6% polyacrylamide gel.