Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site

Abstract

The intergenic region-internal ribosome entry site (IGR-IRES) of dicistroviruses binds to 40S ribosomal subunits in the absence of eukaryotic initiation factors (eIFs). Although the conserved loop sequences in dicistroviral IGR-IRES elements are protected from chemical modifications in the presence of the 40S subunit, molecular components in the 40S subunit, which interacts with the loop sequences in the IRES, have not been identified. Here, a chemical crosslinking study using 4-thiouridine-labeled IGR-IRES revealed interactions of the IGR-IRES with several 40S proteins but not with the 18S rRNA. The strongest crosslinking signal was identified for ribosomal protein S25 (rpS25). rpS25 is known to be a neighbor of rpS5, which has been shown to interact with a related IGR-IRES by cryo-electron microscopy. Crosslinking analysis with site-directed mutants showed that nucleotides UU(6089-6090), which are located in the loop region in conserved domain 2b in the IRES, appear to interact with rpS25. rpS25 is specific to eukaryotes, which explains why there is no recognition of the IGR-IRES by prokaryotic ribosomes. Although the idea that the IGR-IRES element may be a relict of a primitive translation system has been postulated, our experimental data suggest that the IRES has adapted to eukaryotic ribosomal proteins.

Figure 1.

Secondary structure model of the PSIV IGR-IRES, showing nucleotides that are protected from chemical probes in the presence of the 40S ribosomal subunit. Dots and asterisks indicate the base–pair interaction for stems and pseudoknots, respectively. To facilitate description of the IRES structure, the IRES is divided into domain 1, domain 2 and domain 3, as indicated by the dotted lines. Domain 2 is further divided into domains 2a and 2b. Nucleotides that are protected, in the presence of the 40S ribosome, from DMS modification are marked with circles and those from CMCT are marked with squares. Nucleotides that are protected from hydroxyl-radical cleavage are shaded (Modified from Nishiyama et al. (9)).

Figure 2.

Identification of a ribosomal protein crosslinked with s⁴U-labeled PSIV IRES domains 1–2. The origin of the first-dimension gel electrophoresis is indicated by an open triangle. (A) Silver-stained gel after separation of 40S ribosomal proteins. (B) Autoradiograph of a second-dimension gel. The seven signals that were reproducibly detected are indicated by broken squares and numbered from 1 to 7. (C) Autoradiograph of a second-dimension gel containing samples that were incompletely digested with RNase A. The locations of spots of silver-stained proteins are marked with dotted circles. Ribosomal proteins S2, S4, S6, S19, S25 and S26 were identified by mass spectrometry using protein spots excised from a similarly prepared gel.

Figure 3.

Evidence of contact between the s⁴U-labeled IGR-IRES of PSIV and the rpS25 protein of Drosophila. Drosophila 40S ribosomes were mixed with each RNA and irradiated with a near-UV lamp. After partial RNase A digestion, the reaction mixtures were separated on SDS-polyacrylamide (15%) gels and rpS25 proteins were detected by western blot. Lane 1, 40S ribosome with no irradiation; lane 2, 40S ribosome irradiated with unlabeled PSIV IRES; lane 3, 40S ribosome irradiated with s⁴U-labeled PSIV IRES; lane 4, 40S ribosome irradiated with s⁴U-labeled EMCV IRES. The position of the rpS25 protein is marked on the right. Asterisks indicate the positions of crosslinked rpS25 proteins in lane 3.

Figure 4.

Base substitutions in the single-stranded regions of domains 1–2 of the PSIV IRES to identify uridine residues that crosslink to the 40S ribosomal proteins. Mutations indicated by A–I correspond to the mutant IRES elements used in the experiments in Figure 5. To facilitate T7 transcription and HindIII digestion, the 5′ and 3′ terminal sequences were modified from the native sequence shown in Figure 1.

Figure 5.

Identification of uracil residues of the IRES that interact with rpS25. Shown are autoradiographs of second-dimension gels separating the Drosophila 40S ribosomal proteins crosslinked with domain 1–2 mutants of the PSIV IRES containing s⁴U. Signals corresponding to the rpS25 are circled by broken lines. (A) U6012A+U6015A+U6017A mutation; (B) UU6031AA+UUU6036-8AAA mutation; (C) UU6044-5AA mutation; (D) UU6062-3AA+U6066A mutation; (E) UU6073-4AA mutation; (F) UU6082-3CC mutation; (G) UUU6089-91AAA mutation; (H) UU6096-7CC mutation and (I) U6130A mutation.