Abstract
Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.
Highlights
Internal ribosome entry site (IRES) elements promote internal initiation of translation using cap-independent mechanisms (Yamamoto et al, 2017)
The foot-and-mouth disease virus (FMDV) IRES element is organized in domains, designated 1, 2, 3, 4, and 5 (Lozano & Martinez-Salas, 2015)
We describe a robust RNA–protein interaction approach, which allows detecting ribonucleoprotein complexes associated with specific subdomains of the IRES element
Summary
Internal ribosome entry site (IRES) elements promote internal initiation of translation using cap-independent mechanisms (Yamamoto et al, 2017). Despite performing the same function, IRES elements, which were first identified in the RNA genome of picornavirus, are characterized by a high diversity of sequences, secondary structures, and requirement of factors to assemble translation competent complexes, which led to their classification into different types. The picornavirus type II IRES elements such as the encephalomyocarditis (EMCV) and foot-and-mouth disease virus (FMDV) differ in 50% of their primary sequence, yet they fold into similar secondary structures (Lozano & Martinez-Salas, 2015). The basal region of this domain consists of a long stem interrupted with bulges that include several noncanonical base pairs and a helical structure essential for IRES activity. The apical region harbors conserved motifs essential for IRES activity, which mediate tertiary interactions (Fernandez-Miragall & MartinezSalas, 2003; Jung & Schlick, 2013; Lozano et al, 2016). The implication of structural features of domain 3 in the interaction with transacting factors and their potential functions remain poorly studied and need to be investigated
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