BioProject

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. More...

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We use in cell MIME to define RNA elements within the 5’ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identify three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis identifies the 73AAUAAA78 canonical polyadenylation motif within the 5’ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production. Overall design: In cell Mutational Interference Mapping Experiment (in cell MIME) for identifying regulatory RNA controlling HIV-1 replication. Sequencing of input DNA, cellular RNA and viral RNA from two mutant libraries and a WT control (9 samples in total) Less...