Cap‐Independent Translation Initiation Driven by a 13‐nucleotide motif
Authors: Flores Megan A. , Juba Amber N. , Chaput John C. , Wellensiek Brian P. 
Published: June 2021

In eukaryotes, production of proteins is an essential biological process that typically occurs by a mechanism dependent on the 7‐methylguanosine cap at the 5′ end of an mRNA transcript. During normal cellular processes, it is this cap‐dependent (CD) mechanism that synthesizes proteins in three phases: initiation, elongation, and termination. The initiation phase is dependent upon recognition of the cap structure and attachment of the ribosomal pre‐initiation complex (PIC) to facilitate scanning of the mRNA 5′ ‐ untranslated region (UTR) in search for the initiation codon. Translation then proceeds with elongation after the initiation codon is identified. Current research indicates an alternative to the CD translation initiation mechanism, referred to as cap‐independent (CI) translation. In CI translation, recognition of the 5′ cap structure is not required for initiation of translation to occur. Several studies have demonstrated the CI mechanism to function during circumstances including stress conditions of apoptosis, hypoxia, mitosis, or inhibition of the CD mechanism. The literature suggests an internal ribosome entry site (IRES) is one possible mode CI translation can occur. An IRES directly recruits the ribosome to that specific site, regardless of its location within the RNA strand. In efforts to address the uncertainty of IRES occurrences within RNA leader sequences in human transcripts, a recent study revealed a library of human translation enhancing elements (TEEs) that can stimulate the CI mechanism. Detailed comparative functional sequence alignment analysis of the TEEs revealed a common 13‐nucleotide (13mer) motif capable of stimulating translation initiation. In order to evaluate the potential of the 13mer to function as an IRES, a series of RNA leader sequences were constructed in a reporter plasmid system containing a stable hairpin loop in the 5′‐UTR. In this system, the hairpin loop blocks ribosomal scanning due to CD initiation, resulting in reporter expression as evidence of the 13mer functioning as an IRES downstream of the hairpin loop. A series of TEEs were evaluated in this system with and without the 13mer motif. The presence of the 13mer up‐regulated translational activity, however only in certain sequence contexts. The IRES activity of the 13mer was then confirmed in a dual luciferase reporter plasmid (dlp), with the RNA leader sequences located internally between the renilla luciferase and firefly luciferase genes. In addition, translational activity was observed when CD translation was specifically inhibited using a eIF4E/eIF4G‐1 protein interaction inhibitor. Results of this study elucidate the capacity for the CI mode of translation from the human genome by demonstrating IRES activity. The identification of the 13mer as an IRES provides an opportunity to explore the mechanistic details of human IRESs and the contributions to protein production from the human genome.Support or Funding InformationIntramural Midwestern University funds awarded to BPW, and by an NIH grant awarded to JCC (Eureka Award; GM085530)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.