Secondary Structure Prediction for RNA Sequences Including N6-methyladenosine

Abstract

There is increasing interest in the roles played by covalently modified nucleotides in mRNAs and non-coding RNAs. New high-throughput sequencing technologies localize these modifications to exact nucleotide positions. There has been, however, and inability to account for these modifications in secondary structure prediction because of a lack of software tools for handling modifications and a lack of thermodynamic parameters for modifications. Here, we report that we solved these issues for N6-methyladenosine (m6A), for the first time allowing secondary structure prediction for a nucleotide alphabet of A, C, G, U, and m6A. We revised the RNAstructure software package to work with any user-defined alphabet of nucleotides. We also developed a set of nearest neighbor parameters for helices and loops containing m6A, using a set of 45 optical melting experiments. Interestingly, N6-methylation decreases the folding stability of structures with adenosines in the middle of a helix, has little effect on the folding stability of adenosines at the ends of helices, and stabilizes the folding stability for structures with unpaired adenosines stacked on the end of a helix. The parameters were tested against an additional two melting experiments, including a consensus sequence for methylation and an m6A dangling end. The utility of the new software was tested using predictions of the structure of a molecular switch in the MALAT1 lncRNA, for which a conformation change is triggered by methylation. Additionally, human transcriptome-wide calculations for the effect of N6-methylation on the probability of an adenosine being buried in a helix compare favorably with PARS structure mapping data. Now users of RNAstructure are able to develop hypothesis for structure-function relationships for RNAs with m6A, including conformational switching triggered by methylation.