The long noncoding RNA Malat1 contains an internal ribosome entry site mediating micropeptide translation

To determine the structure of the Malat1 IRES, fbDMS-MaP structure probing experiments were performed in BV2 cells for endogenous mouse Malat1 probing, and N2a cells for probing of reporter constructs pDual-GFP-(1-739) IRES-RFP and pDual-GFP-(558-695) IRES-RFP. Two biological replicates were collected for each probing target. For each replicate, fbDMS-MaP includes a sample treated with dimethyl sulfate (modified), and one sample treated with ethanol (unmodified). DMS reacts with the Watson-Crick face of unpaired bases in RNA, covalently modifying them with a methyl adduct. These modifications are encoded as mutations during reverse transcription with MarathonRT. Amplicons are then PCR amplified, next-generation sequencing libraries are prepared, and sequenced using an Illumina NextSeq 2000 platform. The sequencing data are aligned to the reference sequence, and a reactivity profile of 'modified' and 'unmodified' samples is used to calculate the DMS reactivity values of each base. These data can subsequently be used for RNA secondary structure modelling.