In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader

Abstract

Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2′OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.

Structure of the HIV-1 RNA packaging signal

“…S4). These findings support proposals that variations in structure predictions are at least partly due to site-specific structural heterogeneity associated with the monomer-dimer equilibrium (13, 24). …”

“…Recent in-gel chemical probing of resolved monomeric and dimeric leader RNAs (24), and probing studies under solution conditions that favor either the monomeric or dimeric species (25), showed that SD loop residues are relatively unreactive in the dimeric RNA, consistent with the Ψ CES structure. Pseudo-free energy calculations indicate that the in-gel reactivity data for the dimeric leader (24) are in better agreement with the Ψ CES NMR structure than the proposed model (~25% lower experimental pseudo-free energy (18); fig. S4).…”

RiboCAT: a new capillary electrophoresis data analysis tool for nucleic acid probing

“…As a proofof-principle, this new tool was used to analyze SHAPE data collected on the HIV-1 5 ′ -untranslated region (5 ′ UTR). The analyzed data show good agreement with published reactivity data and reproduce the previously determined secondary structure Kenyon et al 2013), but required significantly less user intervention and data analysis time than previous tools.…”

“…Using both NMIA and 1M6, the previous SHAPE-derived secondary structure of the HIV-1 5 ′ UTR Kenyon et al 2013) was replicated using this analysis method. The only variation is the lack of two A-U base pairs toward the top of the poly(A) stem in the prediction based on the 1M6 data.…”

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

“…Other studies indicated that TAR is not essential for genome dimerization and packaging and that mutations that disrupt base pairing in the stem of TAR can lead to aberrant dimerization and packaging (which was attributed to misfolding) (60). On the other hand, chemical probing studies suggested that the base of the TAR hairpin, rather than the Tat-binding bulge or apical loop, may be involved in regulating a monomer-dimer structural switch (61). The present studies provide clear evidence that residues of TAR adopt an intramolecular hairpin structure in the context of the intact 5′-leader.…”