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

Kenyon JC, Prestwood LJ, Le Grice SF, Lever AM - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire, CB2 0QQ, UK and HIV-Drug Resistance Program, Centre for Cancer Research, National Cancer Institute, P.O. Box B, Building 535, Frederick, MD 21702-1201, USA.

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.

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In-gel SHAPE of monomeric and dimeric HIV-1 packaging signal RNA shows significant differences in reactivity between monomer and dimer. (A) Schematic diagram of the HIV-1 RNA examined. Nucleotides are numbered every 50 bases and marked every 10 bases. (B) Ethidium bromide-stained polyacrylamide gel slice showing HIV-1 RNA monomer and dimer excised for probing. (C) NMIA reactivity of monomer (red) and dimer (blue) for nucleotides 100–354. The position of the 6 nt DIS is marked above. Results are an average of 7–10 independent experiments. (D) Plot of average SHAPE reactivity of the monomer subtracted from the average NMIA reactivity of the dimer at each nt position. Colour shows statistical significance by t-test, where purple bars are statistically significant and green are not significant, to P < 0.01.
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gkt690-F2: In-gel SHAPE of monomeric and dimeric HIV-1 packaging signal RNA shows significant differences in reactivity between monomer and dimer. (A) Schematic diagram of the HIV-1 RNA examined. Nucleotides are numbered every 50 bases and marked every 10 bases. (B) Ethidium bromide-stained polyacrylamide gel slice showing HIV-1 RNA monomer and dimer excised for probing. (C) NMIA reactivity of monomer (red) and dimer (blue) for nucleotides 100–354. The position of the 6 nt DIS is marked above. Results are an average of 7–10 independent experiments. (D) Plot of average SHAPE reactivity of the monomer subtracted from the average NMIA reactivity of the dimer at each nt position. Colour shows statistical significance by t-test, where purple bars are statistically significant and green are not significant, to P < 0.01.

Mentions: As in-gel SHAPE can probe individual RNAs of different electrophoretic mobility within a mixed population, it follows that it could be used to examine conformational switches or dimerization processes. The HIV-1 RNA genome dimerizes via palindromic sequences (GCGCGC, GUGCAC or GUGCGC (32)) within the 5′ UTR, designated the DIS. Several structural models have been proposed for the monomeric and dimeric RNAs, often using mutagenesis as a means of isolating and probing the structure of a monomer, rather than by isolating a purely monomeric population of wild-type sequence (20,33). Many studies have concluded that the DIS is exposed in the monomer, and that the overall structure of a monomer is similar to one-half of the dimer (5,17,20). Alternative structural switch models suggest that in the monomer the DIS is paired with a 5′ sequence (21,22). Because in-gel SHAPE distinguishes individual RNA structures without the need for mutagenesis, the wild-type monomeric and dimeric populations were studied. The packaging signal (nts 1–413 of the HIV-1 genome, shown schematically in Figure 2A), prepared by in vitro transcription, was renatured and fractionated by native gel electrophoresis. The stained portion of the gel is shown in Figure 2B. Bands corresponding to the monomer (400 nt) and the dimer (800 nt) are visible. Unstained monomer and dimer were excised and probed with NMIA as described above. These initial renaturation conditions used previously published buffers (34) and were chosen to produce relatively equal amounts of monomer and dimer. SHAPE reactivity data are given in Supplementary Figure S5 and illustrated for nts 100–354 in Figure 2C. Data are an average of 7–13 replicates at each nucleotide position. The experiment was extremely reproducible; each nucleotide showed equivalent NMIA reactivity in >70% of replicates for the monomer and 79% of replicates for the dimer. This corresponded to an average standard deviation of 0.29 for the monomer and 0.22 for the dimer for nucleotides with reactivity <0.9. The standard deviation was higher among the most reactive category of nucleotides.Figure 2.


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

Kenyon JC, Prestwood LJ, Le Grice SF, Lever AM - Nucleic Acids Res. (2013)

In-gel SHAPE of monomeric and dimeric HIV-1 packaging signal RNA shows significant differences in reactivity between monomer and dimer. (A) Schematic diagram of the HIV-1 RNA examined. Nucleotides are numbered every 50 bases and marked every 10 bases. (B) Ethidium bromide-stained polyacrylamide gel slice showing HIV-1 RNA monomer and dimer excised for probing. (C) NMIA reactivity of monomer (red) and dimer (blue) for nucleotides 100–354. The position of the 6 nt DIS is marked above. Results are an average of 7–10 independent experiments. (D) Plot of average SHAPE reactivity of the monomer subtracted from the average NMIA reactivity of the dimer at each nt position. Colour shows statistical significance by t-test, where purple bars are statistically significant and green are not significant, to P < 0.01.
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Related In: Results  -  Collection

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gkt690-F2: In-gel SHAPE of monomeric and dimeric HIV-1 packaging signal RNA shows significant differences in reactivity between monomer and dimer. (A) Schematic diagram of the HIV-1 RNA examined. Nucleotides are numbered every 50 bases and marked every 10 bases. (B) Ethidium bromide-stained polyacrylamide gel slice showing HIV-1 RNA monomer and dimer excised for probing. (C) NMIA reactivity of monomer (red) and dimer (blue) for nucleotides 100–354. The position of the 6 nt DIS is marked above. Results are an average of 7–10 independent experiments. (D) Plot of average SHAPE reactivity of the monomer subtracted from the average NMIA reactivity of the dimer at each nt position. Colour shows statistical significance by t-test, where purple bars are statistically significant and green are not significant, to P < 0.01.
Mentions: As in-gel SHAPE can probe individual RNAs of different electrophoretic mobility within a mixed population, it follows that it could be used to examine conformational switches or dimerization processes. The HIV-1 RNA genome dimerizes via palindromic sequences (GCGCGC, GUGCAC or GUGCGC (32)) within the 5′ UTR, designated the DIS. Several structural models have been proposed for the monomeric and dimeric RNAs, often using mutagenesis as a means of isolating and probing the structure of a monomer, rather than by isolating a purely monomeric population of wild-type sequence (20,33). Many studies have concluded that the DIS is exposed in the monomer, and that the overall structure of a monomer is similar to one-half of the dimer (5,17,20). Alternative structural switch models suggest that in the monomer the DIS is paired with a 5′ sequence (21,22). Because in-gel SHAPE distinguishes individual RNA structures without the need for mutagenesis, the wild-type monomeric and dimeric populations were studied. The packaging signal (nts 1–413 of the HIV-1 genome, shown schematically in Figure 2A), prepared by in vitro transcription, was renatured and fractionated by native gel electrophoresis. The stained portion of the gel is shown in Figure 2B. Bands corresponding to the monomer (400 nt) and the dimer (800 nt) are visible. Unstained monomer and dimer were excised and probed with NMIA as described above. These initial renaturation conditions used previously published buffers (34) and were chosen to produce relatively equal amounts of monomer and dimer. SHAPE reactivity data are given in Supplementary Figure S5 and illustrated for nts 100–354 in Figure 2C. Data are an average of 7–13 replicates at each nucleotide position. The experiment was extremely reproducible; each nucleotide showed equivalent NMIA reactivity in >70% of replicates for the monomer and 79% of replicates for the dimer. This corresponded to an average standard deviation of 0.29 for the monomer and 0.22 for the dimer for nucleotides with reactivity <0.9. The standard deviation was higher among the most reactive category of nucleotides.Figure 2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire, CB2 0QQ, UK and HIV-Drug Resistance Program, Centre for Cancer Research, National Cancer Institute, P.O. Box B, Building 535, Frederick, MD 21702-1201, USA.

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.

Show MeSH