GSE196043
mRNA aging shapes the Cap2 methylome in mammalian mRNA
The mRNA cap structure is a major site of dynamic mRNA methylation.  mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first, or both the first and second transcribed nucleotide, respectively.  However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear.  Here we describe CLAM-Cap-Seq, a method for transcriptome-wide mapping and quantification of Cap2.  We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs.  Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol.  As a result, Cap2 is enriched on long-lived mRNAs.  We find that large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions where RIG-I expression is increased.  The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind and activate RIG-I.  We find that the slow Cap2 methylation rate allows Cap2 to accumulate on host mRNAs yet ensures that low Cap2 levels occur on newly expressed viral RNAs.  Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response. Overall design: We developed next-generation sequencing methods for quantification of Cap2 methylation in bulk mRNA (CapTag-seq), as well as in individual mRNAs in a transcriptome-wide manner (CLAM-Cap-seq). Furthermore, we developed tools for measuring translation and stability of transcript-start nucleotide (TSN) mRNA isoforms by combining polysome profiling and actinomycin D treatment with TSS-seq (transcription start site-sequencing).