Probing the structure of the lncRNA DAMER and its interaction with ATF4 mRNA using SHAPE-MaP
This experiment was conducted to obtain direct structural evidence for the base-pairing interaction between DAMER and ATF4 mRNA. In vitro transcribed RNAs (DAMER-WT, ATF4 mRNA, DAMER mutants) were folded either alone or together and subsequently modified with the SHAPE reagent 1-methyl-7-nitroisatoic anhydride (1M7). A DMSO-only reaction served as the negative control. Sites of modification, which indicate flexible nucleotides, were identified as mutations during reverse transcription. The resulting cDNA was used for library construction and deep sequencing. The data were processed using ShapeMapper2 to calculate reactivity profiles and model RNA secondary structures. Overall design: In vitro transcribed RNA: DAMER (wild-type and mutants ?A4B, ?A5B) and ATF4 mRNA 5' UTR. RNA samples were prepared under different conditions: (1) DAMER alone, (2) ATF4 mRNA alone, (3) DAMER and ATF4 mRNA co-incubated. Each condition was treated with either the SHAPE reagent 1M7 or DMSO as a negative control.