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. 2023 Jul;29(7):865-884.
doi: 10.1261/rna.079375.122. Epub 2023 Apr 6.

Principles, mechanisms, and biological implications of translation termination-reinitiation

Madeline E Sherlock  1 , Laura Baquero Galvis  2 , Quentin Vicens  2   3 , Jeffrey S Kieft  2   3 , Sujatha Jagannathan  1   3
Affiliations

Principles, mechanisms, and biological implications of translation termination-reinitiation

Madeline E Sherlock et al. RNA. 2023 Jul.

Abstract

The gene expression pathway from DNA sequence to functional protein is not as straightforward as simple depictions of the central dogma might suggest. Each step is highly regulated, with complex and only partially understood molecular mechanisms at play. Translation is one step where the "one gene-one protein" paradigm breaks down, as often a single mature eukaryotic mRNA leads to more than one protein product. One way this occurs is through translation reinitiation, in which a ribosome starts making protein from one initiation site, translates until it terminates at a stop codon, but then escapes normal recycling steps and subsequently reinitiates at a different downstream site. This process is now recognized as both important and widespread, but we are only beginning to understand the interplay of factors involved in termination, recycling, and initiation that cause reinitiation events. There appear to be several ways to subvert recycling to achieve productive reinitiation, different types of stresses or signals that trigger this process, and the mechanism may depend in part on where the event occurs in the body of an mRNA. This perspective reviews the unique characteristics and mechanisms of reinitiation events, highlights the similarities and differences between three major scenarios of reinitiation, and raises outstanding questions that are promising avenues for future research.

Keywords: recycling; reinitiation; termination; translation regulation.

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Figures

FIGURE 1.
FIGURE 1.
Comparison of translation termination–reinitiation to other unique types of initiation and translation events. Cartoon representations of ribosomes on mRNAs undergoing (A) reinitiation, (B) intercistronic internal ribosome entry site (IRES)-driven initiation, (C) frameshifting, (D) stop codon readthrough, (E) 2A-mediated peptide bond skipping, and (F) leaky scanning. All possible protein products produced from each transcript are depicted, and the key features, requirements, and outcomes of the scenarios caused by these translation events are summarized on the right.
FIGURE 2.
FIGURE 2.
Characteristics of mRNAs that support translation reinitiation. Different gene organization contexts lead to three reinitiation scenarios. Short upstream open reading frames (uORFs) are the hallmark of scenario 1, with reinitiation occurring in what would otherwise be considered the 5′ untranslated region. A substantially longer first ORF would lead to scenario 2, where reinitiation accesses a downstream ORF (dORF) in what would otherwise be considered the 3′ untranslated region. Scenario 3 happens when reinitiation occurs within an ORF, typically brought on by a premature termination codon. Green: start codon; red: stop codon; black boxes (scenario 2): sequences/structure motifs for termination/reinitiation coupling.
FIGURE 3.
FIGURE 3.
Internal and external factors and features that influence reinitiation mechanisms. (A) Depending on the step at which recycling fails, the 60S subunit may or may not dissociate (mediated by ABCE1 and eIF3j during ribosome recycling) before a reinitiation event occurs. Failure to remove the P-site tRNA and/or the mRNA from the 40S subunit (mediated by eIF2D or DENR/MCT-1 during ribosome recycling) can also lead to reinitiation events. (B) The identity of the amino acids of the nascent peptide from the upstream ORF that are still in the exit channel at the time of termination/failure to recycle may influence reinitiation frequency. (C) Post-transcriptional modifications of the mRNA, such as N6-Methyladenosine (m6A) or pseudouridine (Ψ), can act as a regulatory mechanism to regulate the frequency of reinitiation. (D) Depending on the scenario, the start codon can be upstream, overlapping, or downstream from the first stop codon. The requirements vary greatly between the scenarios of reinitiation and a canonical AUG start codon is not always necessary. (E) The proximity of the stop codon of the upstream ORF with relation to the start codon for the downstream ORF is a major determinant for whether reinitiation occurs. (F) Intramolecular secondary and tertiary structures in the mRNA can influence reinitiation frequency or start codon selection, and in some cases reinitiation is entirely dependent on these structural elements. (G) The set of initiation factors necessary for reinitiation have yet to be determined definitively and likely vary greatly between different scenarios or circumstances of reinitiation. If required, initiation factors either remain bound through the first round of translation or might need to be rerecruited. (H) Met-tRNAi delivery, typically performed by eIF2, could occasionally be carried out by other factors such as eIF2D or DENR/MCT-1 under different conditions and scenarios of reinitiation, although sometimes reinitiation occurs independent of this component.
FIGURE 4.
FIGURE 4.
Functional consequences of translation reinitiation. The three reinitiation scenarios have different outcomes in terms of the functions of encoded proteins. Short uORFs in scenario 1 are considered primarily regulatory, with the act of their translation and not the peptide sequence itself dictating expression of the main ORF, which has critical cellular functions. Scenario 2 produces two functional proteins at a precise stoichiometric ratio, which so far has mainly been observed in viruses. It is unclear what, if any, roles the peptide sequences encoded by downstream ORFs play in eukaryotic genes. Scenario 3 can have different consequences—neutral, beneficial, or detrimental—depending on the position of the PTC and the nature of the two protein products that would be translated as a single protein in the absence of a nonsense mutation. Green: start codon; red: stop codon; black open boxes (scenario 2): sequences/structure motifs for termination/reinitiation coupling.
FIGURE 5.
FIGURE 5.
Evolution and therapeutic applications of translation reinitiation. (A) Sequences or structures can evolve in the viral genome to promote expression of different or new open reading frames (scenario 2). uORFs can be created via mutations or transposon insertion (scenario 1). The acquisition of a downstream AUG can allow an otherwise detrimental PTC to be tolerated (scenario 3). (B) Small molecule or RNA-based therapeutics targeting RNA sequences, RNA structures, or protein factors that impact reinitiation could be developed to inhibit viral proliferation or alleviation of a genetic disease due to acquisition of an upstream AUG or a PTC. Green: start codon; red: stop codon; black boxes: sequences/structure motifs for termination/reinitiation coupling.
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