A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Abstract

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.

Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

“…Another possibility is applying a spheroplasting-based cell lysis approach, which requires enzymatic digestion of the cell wall followed by lysis using osmotic pressure, freeze-thawing, or other disrupting strategies (Darling et al 1969;Mann et al 1972;Izawa and Unger 2017). Finally, the cryogenic lysis technique that has proven to be a very powerful approach for maintaining translational activity (Trainor et al 2021) can be used.…”

“…In our previous work, we have developed, validated, and applied a cell-free translation system (CFE) that represents a translationally competent yeast lysate capable of protein synthesis from an mRNA reporter or endogenous cellular transcripts (Trainor et al 2021). Although this system allows us to address roles of cis and trans factors during protein synthesis, as exemplified in (Trainor et al 2021), manipulating ribosomes exclusively to research effects of ribosomedirected stress on translation remains elusive.…”

“…PCR reactions and reporter RNA synthesis were performed as described in (Trainor et al 2021) with minor modifications. Briefly, a sequence corresponding to a reporter gene cloned in pYes2 as described in the "Plasmids" section was amplified with DreamTaq polymerase using forward (5'-CGGATCGGACTACTAGCAGCTG-3') and reverse (5'-TTCATTAATGCAGGGCC GCAAATT-3') primers that anneal upstream and downstream of the T7 promoter and the CYC1 terminator elements on pYes2, respectively.…”

“…Protein pellets were analyzed as described in (Trainor et al 2021). In brief, protein pellets were resuspended in 50 l of 1 x SDS-PAGE loading dye, 10 L were resolved by SDS-PAGE using 10% polyacrylamide gels, transferred onto nitrocellulose membrane, and blocked with 10% milk in PBS-0.1% Tween 20 (PBST).…”

Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

“…To analyze in vitro translation reaction products, proteins were isolated from 15 L of the translation reactions using TRI REAGENT-LS according to the manufacturer's recommendations. Protein pellets were analyzed as described in (Trainor et al 2021a). In brief, protein pellets were resuspended in 50 L of 1 x SDS-PAGE loading dye, 10 L were resolved by SDS-PAGE using 10% polyacrylamide gels, transferred onto nitrocellulose membrane, and blocked with 10% milk in PBS-0.1% Tween 20 (PBST).…”