Abstract
RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.
Highlights
RNA-protein interactions govern many viral and host cell processes
Previous studies that featured cross-linking of unmodified RNA to proteins have identified a minimal number of binding sites[16,21,37]
By applying capillary sequencing technology with appropriate controls, and combining this with a thorough secondary structural analysis, we show that it is possible to use XL-SHAPE to scan a large RNA, identifying multiple areas of RNA-protein interaction and concurrent structural change
Summary
RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNAprotein interaction and sites of RNA structural remodelling. A powerful secondary structure probing method (SHAPE- selective 2′ OH acylation analyzed by primer extension) with a cross-linking technique would provide a more comprehensive picture of RNA-protein interactions We used this technique to gain new insight into HIV-1 genome packaging. The viral genome is a single-stranded RNA molecule that dimerizes via a palindromic site in the 5′ leader[7,8] This region, along with the beginning of the gag gene, is known as the packaging signal (psi)[9,10]. One stem-loop is known to bind Gag with high affinity (SL3), the initial capture is believed to involve a small number of Gag proteins binding to psi[11] Analyzing this interaction is complicated because the RNA changes structure upon dimerization, exposing different regions that are potential additional protein binding sites[12,13]. The bulky adduct blocks reverse transcription; termination products can be electrophoresed alongside sequencing ladders to quantitate the relative amounts of SHAPE adduct at each nucleotide
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