Full-length sense and antisense TROJAN strands were cloned into pGEM-T Easy Vector (Promega). In vitro transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs), and RNA was purified using the RNeasy MinElute Cleanup Kit (QIAGEN). TROJAN was labeled using the Biotin 3′ End DNA Labeling Kit (Thermo Fisher Scientific). Cells were harvested and resuspended in freshly prepared radioimmunoprecipitation assay lysis buffer supplemented with RNaseOUT recombinant ribonuclease inhibitor (50 U/ml; Thermo Fisher Scientific), SUPERase In RNase inhibitor (50 U/ml; Thermo Fisher Scientific), and a protease/phosphatase inhibitor cocktail (Roche). RNA secondary structure formation was assessed as follows. Briefly, 20 μg of total biotin-labeled RNA was first restructured in RNA structure buffer [10 mM tris (pH 7.0), 0.1 M KCl, and 10 mM MgCl2] at 90°C for 2 min, immediately placed on ice for 2 min, and then incubated at room temperature for 20 min. Labeled RNA was incubated with streptavidin magnetic beads (Pierce) for 30 min at room temperature with agitation. The RNA-captured magnetic beads were washed twice with wash buffer (Thermo Fisher Scientific). In total, 10 mg of SILAC cell lysates was added to yeast transfer RNA (0.1 μg/μl) and 5 mM MgCl2. Sense and antisense RNA captured on the magnetic beads, as well as blank beads, were incubated with Lys0 and Arg0, Lys4 and Arg6, and Lys8 and Arg10 cell lysates, respectively, in protein-RNA binding buffer (Thermo Fisher Scientific) overnight at 4°C with agitation. RNA binding protein complexes were washed five times with ice-cold wash buffer and boiled in SDS lysis buffer.

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