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

Abstract

Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro, and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.

Rab1b and ARF5 are novel RNA-binding proteins involved in IRES-driven RNA localization

“…The IRES-driven RNA clustering is in agreement with long-range RNA-RNA interactions involving domain 3 (Diaz-Toledano et al, 2017; Ramos & Martinez-Salas, 1999), which could contribute to hold IRES-containing transcripts in specific subcellular location. Furthermore, our data are also in accordance with a recent report showing that IRES-containing mRNAs are enriched in ribosomal subunits purified from cell lysates relative to cap-mRNAs (Lozano et al, 2018). Interestingly, purified ribosomes induced SHAPE reactivity changes within domains 2 and 3 of the IRES, including the apical SL3abc subdomain.…”

“…Cells transfected with constructs expressing cap-luc or IRES-luc mRNA were first used to determine the expression of the reporter protein. As expected, both RNAs produced luciferase activity although to different extent ( Fig 5A) (Lozano et al, 2018).…”

“…For SL3a, oligonucleotides were annealed and inserted into pBSMrnaStrep via SalI and AatII. Constructs pIRES-luc and pCAP-luc, tagged with MS2 hairpins, were described (Lozano et al, 2018). Plasmid peGFP-C1-Rab1b was generated by PCR using primers C1-GFPRabs, C1-GFPRabas, and template pPB-N-His-Rab1b.…”

Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

“…b) Summary of Gemin5 binding to the ribosome and L3/L4 ribosomal proteins, obtained with the wild type (amino acids 1–739) or the indicated mutants. Functional analysis of Gemin5 role on translation measured in HEK293 cell lysates expressing the full‐length protein Xpress‐G5, or the indicated mutants, and the reporter cap‐Luc mRNA . Values (mean ± SEM) are normalized to cells expressing the empty vector (control).…”

A novel dimerization module in Gemin5 is critical for protein recruitment and translation control

“…HEK293 cell monolayers (2x10 5 ) were cotransfected with a plasmid expressing luciferase in cap-dependent or IRES-dependent manner (pCAP-luc, pIRES-luc) (Lozano et al, 2018), and a plasmid expressing Xpress-p85-wt, Xpress-p85-A951E, Xpress-p85∆RBS1/2, or the corresponding empty vector side by side using lipofectamine LTX (Thermo Scientific).…”