Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16

Therapeutic mRNAs are generated using modified nucleotides, namely N 1-methylpseudouridine (m1W) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple- stranded RNA, globally modified with N 6-methyladenosine (m6A), pseudouridine (W), or m1W. UV thermal denaturation assays showed that m6A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by X 20 degrees C, W stabilizes the Hoogsteen and Watson-Crick faces of the RNA by X 12 degrees C, and m1W has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by X 9 degrees C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m6A, W , or m1W. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means.