Mapping and Direct Sequencing of the Non-Canonical Cap Code

Mapping and Direct Sequencing of the Non-Canonical Cap Code

From their creation to their degradation, mRNAs are under extensive regulation. This regulation is directed not only by their nucleotide sequence and their promoter but also by post- and co-transcriptional modifications attached to the RNA. One of the most ubiquitous of these modifications in eukaryotes is the 5' cap — typically a methylated guanine (m7G) attached inversely to the first transcribed nucleotide. Several decades of research have revealed the m7G cap as an interface for regulating stability, splicing, polyadenylation, localization, and translation. The m7G cap was long presumed to be the only functional cap in eukaryotes, but this view was recently overturned by the discovery of the non-canonical nicotinamide adenine dinucleotide (NAD+) cap. Following this, several other eukaryotic cap structures were discovered: NADH, flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The function of most non-canonical caps is unknown, but their presence suggests the existence of a regulatory “cap code”. Currently, the complete cap structure can only be identified through bulk methods that cleave caps from their transcripts, thereby losing the link between mRNAs and their specific caps. For NAD-caps enrichment techniques exist that can be followed by sequencing, but no such method exists for other non-canonical caps, and no sequencing method exists that can identify more than one cap. This limitation is a significant obstacle in understanding the cap code and the function of its more enigmatic members. The objective of this project is to 1) develop a novel single-molecule method that can directly sequence native caps with their full-length RNA transcripts, 2) use this method to characterize the cap-ome across tissues and development, and 3) use the resulting temporal and spatial atlas of capped RNAs to determine the function of non-canonical caps and understand the non-canonical cap code