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

BACKGROUND: 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. RESULTS: Here, we perform in vivo selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical profiling on Arabidopsis and generate the in vivo nuclear RNA structure landscape. We find that nuclear mRNAs fold differently from cytosolic mRNAs across translation start and stop sites. Notably, we discover 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. The regulatory role of this RNA structure feature is further confirmed by experimental validation. Moreover, we find the single-strandedness of branch sites is also associated with 3′ splice site recognition. We also identify an RNA structure feature comprising two close-by single-stranded regions that is specifically associated with both polyadenylation and alternative polyadenylation events. CONCLUSIONS: We successfully identify pre-mRNA structure features associated with splicing and polyadenylation at whole-genome scale and validate an RNA structure feature which can regulate splicing. Our study unveils a new RNA structure regulatory mechanism for mRNA processing.