Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE

Figures

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  Figure 1. Conformational changes in the RNA local flexibility of the IRES incubated with native ribosomes. (A) Reactivity patterns for IA (black) and 1M6 (gray) reagents obtained after QuSHAPE software processing for representative regions of domain 2, 3 and 4–5 of the free IRES element. Black and grey arrows depict some examples of enhanced reactivity toward IA or 1M6, respectively. (B) Statistically significant SHAPE differences towards IA and 1M6 of the RNA incubated with R fraction relative to free RNA as a function of the nucleotide position. Red or blue bars depict nucleotides with p-values < 0.05 and absolute SHAPE reactivity differences higher or lower than 0.2, respectively. Grey bars depict non-statistically significant differences (p-values > 0.05 and/or absolute differences D <0.2).

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  Figure 2. Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES-interacting proteins PTB, Ebp1 and Gemin5. This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).

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  Figure 3. RNA structure modeling of the IRES region. Predicted 3D structure models for the IRES imposing SHAPE reactivity values obtained for the free RNA (A) and upon incubation of the IRES transcript with the R extract (B). Domains 2, 3, 4, and 5, subdomains J and K of domain 4, as well as the GNRA tetraloop, loops, and bulges referred to as in the text are indicated. Detailed structure model of the apical region of domain 3 for the free RNA (C), and incubated with R extract (D).

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  Figure 4. Modification of the RNA local flexibility of the IRES incubated with salt-washed ribosomes. (A) Statistically significant SHAPE differences towards 1M6 and IA reagents of the RNA incubated with RSW fraction relative to free RNA as a function of the nucleotide position. (B) Statistically significant SHAPE differences towards IA of the RNA incubated with F, or RSW + F fractions relative to free RNA as a function of the nucleotide position. Symbols as in Fig. 1B.

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  Figure 5. Conformational changes in the local flexibility of the IRES incubated with 40S or 60S ribosomal subunits. (A) Preparation of 40S and 60S ribosomal subunits from HEK293 cells. S30 extracts prepared with high salt buffer were loaded in continuous sucrose gradients and ultracentrifuged to separate the fractions corresponding to 40S and 60S peaks. These fractions were ultracentrifuged to obtain the purified ribosomal subunits. Significant SHAPE differences towards 1M6 and IA of the RNA incubated with 40S subunits (B) or 60S (C) relative to free RNA as a function of the nucleotide position. Symbols as in Fig. 1B.

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  Figure 6. Association of IRES-RNA and cap-RNA to ribosomal subunits in whole cell lysates. (A) Schematic of transcripts cap-luc and IRES-luc. (B) Quantification of IRES-RNA levels (black bar) relative to cap-RNA (white bar) present in total cell lysates monitored by RT-qPCR. (C) Quantification of IRES-RNA relative to cap-RNA levels associated to 40S and 60S subunits. Values represent the mean ± SD obtained in two independent assays. Asterisks denote p-values (*p< 0.05).

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  Figure 7. RNA dynamics of the IRES upon incubation with ribosome fractions. Differential SHAPE reactivity as a function of the nucleotide position depicting IA-1M6 and 1M7-1M6 statistically significant differences obtained for the free RNA (A). The secondary structure of the IRES shows IA enhanced reactivity (slow, pink) and 1M6 enhanced reactivity (stacking, green) (B). Nucleotides are numbered every 50 positions, dots mark every 10 positions. (C,E) Differential SHAPE reactivity for IRES-R complexes, or IRES-RSW complexes, and (D,F) changes in RNA dynamics for IRES-R, or IRES-RSW. Asterisks depict positions lacking differential reactivity relative to the free RNA.

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  Figure 8. Schematic representation of the changes in conformational flexibility and RNA dynamics of the IRES in the presence of the indicated ribosome fractions relative to free RNA.