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1.
FIGURE 1.

FIGURE 1. From: Structural architecture of an RNA that competitively inhibits RNase L.

Secondary structure of the ciRNA. At top is a diagram of the poliovirus RNA genome with open reading frames for the viral protein products shown. The ciRNA is found in the 3CPro coding region. (Below) The proposed secondary structure is shown. Regions in gray are not part of the “minimum” functional ciRNA. Various secondary structure elements are colored and labeled, and the kissing interaction is shown with a dashed line and arrows. The WT ciRNA used in this study did not contain any of the RNA in gray.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.
2.
FIGURE 5.

FIGURE 5. From: Structural architecture of an RNA that competitively inhibits RNase L.

Ab initio shape reconstructions of WT ciRNA from SAXS. (A) Kratky plots constructed from SAXS data of the ciRNA (black), phenyalanine yeast tRNA (red), and the BMV TLS RNA (green). The tRNA and BMV data and curves were previously published (Hammond et al. 2009). (B) Plots of the pairwise distribution functions for the same RNAs shown in panel A. Colors match panel A. (C) Six independent representative shape reconstructions. (D) Average of 10 independent reconstructions.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.
3.
FIGURE 3.

FIGURE 3. From: Structural architecture of an RNA that competitively inhibits RNase L.

Function and structural role of the putative loop E motif. (A) Secondary structure diagram of the putative loop E motif, with noncanonical base-pairs shown. Nucleotides previously mutated and studied are circled in gray; mutations we generated and studied are circled in black, and the mutation is indicated. (B) Bar graph of in vitro cleavage assays with the loop E mutants, WT, and control RNAs. Graph depicts the fluorescence at time = 60 min, averaged from three experiments. Error bars, 1 SD from the mean. (C) Native gels of WT ciRNA and three loop E mutants. The top gel contained 5 mM MgCl2; the bottom contained 2 mM EDTA. (D) Thermal denaturing curves of WT ciRNA and the three loop E mutants. At top is the raw data normalized to reflect fraction RNA folded as a function of temperature; at bottom is the first derivative of the data. The two transitions discussed in the text are indicated.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.
4.
FIGURE 4.

FIGURE 4. From: Structural architecture of an RNA that competitively inhibits RNase L.

Hydroxyl radical (•OH) probing of the WT ciRNA. (A) Representative gel from a •OH probing experiment (using Fe(II)-EDTA) of the WT ciRNA shown in panel C. Two different gel run times are shown. From left to right, the lanes contain an RNase T1 sequencing reaction, a base hydrolysis reaction, the •OH probing in the absence of Mg2+, and the probing in the presence of 10 mM Mg2+. Secondary structural regions are indicated to the right of the gel in colors that match those in Figure 1 and panel C. The dashed box indicates the portion of the gels shown in panel B. (B) Quantification of the portion of the gel shown in panel A with a dashed box, normalized and graphed to show intensity of radiation as a function of location in the gel. The trace in the absence of Mg2+ is blue and in the presence of 10 mM Mg2+ is in green, and the difference is in red. Shaded gray regions indicate regions that show subtle protection in the presence of Mg2+. The trace of the RNase T1 and base hydrolysis lanes are shown in black and gray at the bottom of the graph, respectively. Positions of secondary structure elements are shown under the graph in colors that match panel C. (C) Secondary structure diagram of the WT ciRNA used in the probing, color-coded as in Figure 1. Regions that showed reproducible protection from solvent in three independent probing experiments are shaded dark gray.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.
5.
FIGURE 6.

FIGURE 6. From: Structural architecture of an RNA that competitively inhibits RNase L.

Model of the global architecture of the ciRNA. (A) At left is the proposed secondary structure of the WT ciRNA with elements color coded and labeled. The lengths of several helices in the structure (in term of base-pairs) is indicated. To the right of the secondary structure diagram is a model of the global architecture of the ciRNA, with cylinders representing the helical regions, black lines indicating junction regions, base-pairs that form the kissing interactions shown with gray lines, and L1 and L4 labeled. Hypothetical interactions between the kissing loops and the loop E motif (cyan) are shown with dashed arrows and a question mark. (B) Interaction between the loop E motif found in helix 11 of 23S rRNA from E. coli (blue and red) and other parts of the rRNA (yellow and green) (Zhang et al. 2009). The secondary structure of the loop E motif is shown alongside. Two adenosines in the loop E (red) make contact through the minor groove (arrows) to a G nucleotide (green). (C) At left is the NMR structure of a kissing loop interaction based on the HIV-2 Tar RNA that contains six paired bases in the loop (Chang and Tinoco 1997). At right is the loop E motif from panel B. The dashed arrow indicates a speculative interaction that could form between RNA motifs of this type.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.
6.
FIGURE 2.

FIGURE 2. From: Structural architecture of an RNA that competitively inhibits RNase L.

Function and structural role of the kissing hairpins. (A) Diagram of the mutants used to study the structural role of the kissing hairpin interaction. For clarity, only the L1 and L4 elements are shown. The loop(s) mutated in each RNA are shaded gray. (B, left) Time course of fluorescence from a representative in vitro RNase L cleavage assay. Controls with no added RNA, with a negative control RNA (2123), or without 2-5A activator are shown. The inhibition effect of WT ciRNA also is included. (Right) Comparison of WT, cL1, cL4, and cL1+cL4 mutant RNAs in the cleavage assay. All concentrations of RNA = 200 nM. (C, left) Bar graph depiction of the experiment shown in panel B. The amount of fluorescence at time = 60 min is shown for each experiment. An additional negative control RNA (BMV TLS, a structured RNA form the 3′ UTR of the brome mosaic virus genomic RNA) also is included. One representative experiment is shown. (Right) Bar graph of an in vitro cleavage assay done at elevated RNA concentrations (6 μM) showing how the mutants inhibit nearly as well as WT at this concentration of RNA. Data are three averaged experiments; error bars, 1 SD from the mean. (D) Native gels of WT ciRNA and the cL1, cL4, and cL1+cL4 mutants. The top gel contained 5 mM MgCl2; the bottom contained 2 mM EDTA. (E) Thermal denaturing curves of WT ciRNA and the three kissing loop mutants. At top is the raw data normalized to reflect fraction RNA folded as a function of temperature; at bottom is the first derivative of the data. The two transitions discussed in the text are indicated.

Amanda Y. Keel, et al. RNA. 2012 January;18(1):88-99.

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