In vivo nuclear RNA structurome reveals RNA-structure regulation of mRNA processing in plants

mRNA processing is critical for gene expression. A challenge in regulating mRNA processing is how to recognize the actual mRNA processing sites, such as splice and polyadenylation sites, when the sequence content is insufficient for this purpose. Previous studies suggested that RNA structure affects mRNA processing. However, the regulatory role of RNA structure in mRNA processing remains unclear. Here, we performed in vivo selective 2-hydroxyl acylation analysed by primer extension (SHAPE) chemical profiling on Arabidopsis nuclear RNAs and generated the in vivo nuclear RNA structure landscape. We found that nuclear mRNAs fold differently from cytosolic mRNAs. Notably, we discovered a two-nucleotide single-stranded RNA structure feature upstream of 5 splice sites that is strongly associated with splicing and the selection of alternative 5 splice sites. Moreover, we found the single-strandedness of branch point is also associated with 3 splice site recognition. We also identified an RNA structure feature comprising two close-by single-stranded regions that is specifically associated with both polyadenylation and alternative polyadenylation events. Our work demonstrates an RNA structure regulatory mechanism for mRNA processing.