RNA-guided assembly of Rev-RRE nuclear export complexes
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 2 supplements
HIV RRE RNA adopts a pre-organized compact fold.
(A) SHAPE-based secondary structure of the RRE RNA. Red, orange, and blue dots highlight nucleotides with high, medium, and low SHAPE reactivity, respectively. Nucleotides with no SHAPE reactivity …
Figure 1—figure supplement 1
Designed oligonucleotides can invade and hybridize to the RRE RNA at specific sites.
(A) EMSAs showing AS 100-113 and AS 54-84 can form complexes with the RRE RNA, while shorter oligonucleotides targeting the Stem I region cannot bind to the RRE. (B) Toe-printing assays indicate …
Figure 1—figure supplement 2
SHAPE changes induced by oligonucleotides interactions.
To make this plot, any SHAPE value >1.0 was set to 1.0. This is because changes for any SHAPE reactivities beyond that is not relevant as those positions will be considered highly reactive in both …
Figure 2 with 1 supplement
Model of the compact RRE RNA conformation.
(A) RRE-oligo complexes show scattering patterns different from that of the RRE RNA alone. (B) Porod-Debye plot of RRE RNA and RNA-oligo complexes indicates the native RNA is more folded and the …
Figure 2—figure supplement 1
Guinier plots of the SAXS data.
Left, Guinier plot of the full-length RRE; middle, Guinier plot of the RRE with antisense oligonucleotide 54-84; Right, Guinier plot of the RRE with antisense oligonucleotide 100-113. Guinier …
Figure 3 with 1 supplement
Thermodynamic studies on the Rev-RRE assembly pathway.
(A) SHAPE profiles from samples at different Rev:RRE ratio. The positions showing increase/decrease of SHAPE reactivity are indicated with arrows at the bottom of the plots. (B) Trend of emergence …
Figure 3—figure supplement 1
SHAPE signatures generated by EMSA.
(A) EMSAs for quantification of different Rev-RRE intermediate states. (B) SHAPE signatures calculated based on EMSAs results. Left, SHAPE signatures representing nucleotides with increased SHAPE …
Figure 4 with 2 supplements
Dynamic assembly of the Rev-RRE RNP.
(A) SHAPE profiles for second-resolution snapshots of the RRE at different time points over the course of Rev-RRE assembly. The positions showing increase or decrease of SHAPE reactivity are …
Figure 4—figure supplement 1
Quality and reproducibility test for the SHAPE-Seq data.
(A) Quality statistics for SHAPE-Seq reads used for analysis. Index1 and index2 are two barcodes introduced during the PCR amplification step. R1 and R2 represent the paired-end reads from both …
Figure 4—figure supplement 2
Rev-RRE assembly process exhibits two-step features.
(A) Dendrogram of the rates for SHAPE-reactivity changes at different nucleotides. All the fast-reacting and slow-reacting nucleotides are colored in the same way as in Figure 4, while intermediate …
Figure 5
Functional importance of the Region3.
(A) Table showing the mutations used in this figure. (B) Normalized SHAPE change at 1, 6 and 11 s after Rev binding. The ∼354-nt RRE is shown in black, Region3 mut1 is shown in blue, mut2 is shown …
Figure 6
Model for pre-organized RRE RNA guides sequential binding of Rev to form the Rev-RRE RNP.
RRE RNA forms a compact fold in the absence of Rev. Rev assembly on the RRE starts from a single nucleation point. Region1 and Region2 binding are coupled and the four-Rev complex state can serve as …
Additional files
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Supplementary file 1
Comparison of RRE alone and RRE in complex with antisense oligonucleotides.
- https://doi.org/10.7554/eLife.03656.015
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Supplementary file 2
SHAPE-changing rate demonstrating half-life at different nucleotides. For positions with complex SHAPE reactivity changes, only the six earlier data points were used for the fitting. Data was fitted to one-phase decay/association with automatic outlier elimination using GraphPad Prism 5.0b for Mac OS X.
- https://doi.org/10.7554/eLife.03656.016
