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. 2011 Jul;39(12):5264-75.
doi: 10.1093/nar/gkr114. Epub 2011 Mar 4.

Structural Basis for the Binding of IRES RNAs to the Head of the Ribosomal 40S Subunit

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Free PMC article

Structural Basis for the Binding of IRES RNAs to the Head of the Ribosomal 40S Subunit

Margarita Muhs et al. Nucleic Acids Res. .
Free PMC article

Abstract

Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.

Figures

Figure 1.
Figure 1.
The cryo-EM maps from the wild-type and rpS25-deletion mutant. (a and b) 80S ribosomes from wild-type and mutant, respectively, seen from the L1 side. (c–f) The corresponding 60S (blue, c and d) and 40S (yellow, e and f) subunits from the interface side. Landmarks of the 40S subunit are head (h), body (b), beak (bk), platform (pt), left and right foot (lf and rf, respectively). Landmarks for the 60S subunit are central protuberance (CP), L1 protuberance and stalk base (SB). The arrow at the 40S subunit points the estimated localization of the protein rpS25.
Figure 2.
Figure 2.
Superposition of the 40S subunits from wild-type and the rpS25-deletion mutant. (a) The deletion (yellow) and the wild-type ribosome (green) is shown from the interface side. (b) The difference map (red) between the wild-type and mutant reconstruction shows two peaks at the head of the ΔrpS25 subunit. The most part of the beak side difference overlaps with density in the mutant ribosome, i.e. density is present but weaker than in the control indicative for higher flexibility of this region. In contrast, the difference above the body platform is due to a hole in the density of mutant ribosome indicating the position of the deleted rpS25.
Figure 3.
Figure 3.
Binding and translation efficiency of IRES RNA. (a) Binding of 32P-labelled IRES RNA (domains 1–3) to yeast wild-type (W303) and ΔS25 80S ribosomes. The fraction of IRES RNA bound is the ratio of [32P]RNA retained on the filter to that of the input [32P]RNA. (b) Luciferase activity of IRES-Fluc with lysates; standard deviations are indicated. The Fluc coding region was preceded by 48 nts of the PSIV capsid coding region in order to increase the Fluc activity [see ref. (45)]. (c) His-tag rpS25 or GroES pull down-assay against RNAs. Control lanes 1 and 2: 25 pmol of IRES RNA domains 1 and 2; and 25 pmol of E. coli tRNAPhe, respectively. RNAs co-precipitated by rpS25 (lanes 3 and 4) and GroES (lanes 5 and 6).
Figure 4.
Figure 4.
Footprint experiments showing protected bases of PSIV-IRES by the binding of isolated rpS25A. (a) To detect protected bases, CMCT (lanes 1–4) and RNase T1 (lanes 5–8) treated RNAs were analysed by primer extension. Lanes 1 and 5, no rpS25; lanes 2 and 6, 5-fold molar excess rpS25A over PSIV-IRES; lanes 3 and 7, 10-fold molar excess rpS25A; lanes 4 and 8, 20-fold molar excess rpS25A. (b) Secondary structure of PSIV IRES; (filled square), bases protected against CMCT by rpS25; (filled circle), bases protected against RNase T1.
Figure 5.
Figure 5.
Tertiary-structure model of rpS25 (comprising residues 42–108) together with yeast 80S•CrPV map at 6.7Å resolution (M. Muhs and C. M. T. Spahn, unpublished). (a) Perspective from the intersubunit space. (b) The C-terminus shows possible interactions of rpS25 with helix 41 (h41) of the 18S rRNA. It is flanked by rpS5. H2 is cut by the clipping plane and is not visible here. (c) The two helices H1 and H3 form the core of a HTH motif. The highly conserved residues Arg58 and Arg68 fit into two well-defined density regions bridging rpS25 to the stem loop SL2.3 of the CrPV IRES. (d) At lower thresholds, the cryo-EM density map indicates further interactions between the CrPV IRES and the ribosome (black arrows). The N-terminus (N) is truncated, because it could not be modelled based on our template structure.
Figure 6.
Figure 6.
rpS25 with neighbouring proteins and 18S rRNA. rps25 (red) is shown from the side of the N-terminal helix (H1). It is flanked by ribosomal proteins S18 (left) and S5 (right). The 18S rRNA is depicted in yellow.
Figure 7.
Figure 7.
Interactions of the IRES RNA with the ribosome. (a) Molecular interactions of the CrPV IRES (magenta ribbon) with ribosomal proteins S5 (orange) and S25 (red). (b) A close-up of the interaction partners docked into the cryo-EM density (grey mesh) of the yeast 80S•CrPV complex [(34); M. Muhs and C. M. T. Spahn, unpublished data].

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