Nano-DMS-MaP enables isoform-resolved RNA structure determination
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HIV-1 is a retrovirus that packages an RNA genome, which upon cell entry is reverse transcribed and integrated into the host cell genome. Newly transcribed HIV RNA from this proviral DNA may then be spliced into multiple different transcripts to express viral proteins, or be packaged into the viral genome. Intriguingly, while all HIV RNAs share the majority of the 5' UTR, which contains a major packaging motif, only the unspliced genomic RNA is efficiently packaged into virions.
We therefore hypothesized that structural differences play a major role in regulating this packaging activity.
Current RNA structural probing methods, such as DMS-mutational profiling sequencing (DMS-MaP), use NGS technologies whose short reads cannot be reliably mapped to specific transcripts for shared regions of transcript isoforms. To circumvent this issue we developed and benchmarked Nanopore-based DMS mutational profiling (Nano-DMS-MaP) to simultaneously investigate RNA structures HIV-1 spliced and unspliced transcripts in cells. Our results demonstrate that HIV-1 splice variants have distinct structures that cluster with acceptor site usage. Importantly, our data suggest that in addition to increasing protein diversity, alternative splicing also resultsv in the generation of RNA transcripts with distinct functions through altered RNA structures.