Unique Structures in the 3′ UTR of Blackcurrant Reversion Nepovirus Genomic RNA 1 Promote Translation Initiation

Abstract

Canonical translation initiation is promoted and regulated by an interaction between proteins bound to a cap structure (a methylated guanosine triphosphate, m7G, as the 5′ nucleotide) at the 5′ end and those bound to a polyadenylate [poly(A)] tail at the 3′ end of a messenger RNA (mRNA). This end‐to‐end interaction contributes to the recruitment, positioning, and activation of the translation machinery. Blackcurrant reversion nepovirus (BRV) has two bipartite genomic RNAs (RNA 1 and RNA 2) that do not contain 5′ cap structures, but do have 3′ poly(A) tails. Hence, the mechanism of end‐to‐end interaction in BRV has not been clearly defined. In other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 5′ and 3′ untranslated regions (UTRs) bind via proposed RNA‐RNA kissing‐loop interactions between a 5′ hairpin loop and a 3′ cap independent translation enhancer (CITE). Likewise, the mechanism by which the 5′ and 3′ UTRs recruit protein synthesis machinery is directly related to their primary, secondary and higher‐order structure. Considering that the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our main goal focuses on determining the secondary and possible tertiary structures of the 3′ CITE structure in the genomic RNAs found in the BRV. Toward that goal, we have in vitro transcribed the minimal BRV RNA 1 CITE sequence, and probed our purified RNA with a series of covalent nucleobase, sugar and backbone modifiers. Combining our results, we have found a unique secondary structure for the BRV RNA 1 CITE that differs from a previously proposed structure based upon in silico thermodynamic folding predictions. This structure likely plays an important role regulating protein synthesis of viral proteins encoded by the RNA 1 message by either facilitating end‐to‐end interaction with the 5′ UTR and/or directly recruiting important translation protein machinery.Support or Funding InformationMSU Denver College of Letters, Arts & Sciences Mini‐grant, MSU Denver Undergraduate Research Grant, MSU Denver Center for Advanced STEM Education Undergraduate Research Grant