We ARE boosting translation: AU-rich elements for enhanced therapeutic mRNA translation

Introduction

Messenger RNA (mRNA) therapeutics has emerged as a transformative approach for treating a range of diseases, from genetic disorders to cancers and infectious diseases.

Unlike traditional protein-based drugs that are administered in a “ready-to-use” form, mRNA therapies are administered as a pro-drug where the pharmacologically active molecule is the product of its translation.

Efficient or prolonged translation of mRNA is crucial for the success of RNA-based therapeutics. The amount of protein produced from mRNA molecules determines the therapeutic efficacy of the treatment. Low translation efficiency can lead to ineffective therapies, requiring higher mRNA doses and increasing the risk of adverse effects. Also, optimizing mRNA translation could allow dose-sparing strategies which can results in increase vaccine availability to address the global crisis during pandemics. Well-established strategies of sequence optimization include Kozak and codon usage optimization, dsRNA avoidance, and polyA length.

An approach to prolong RNA translation relies on two different biotechnologically implemented classes of RNAs namely circular RNAs (circRNAs) and self-amplifying RNAs (saRNAs). circRNAs are characterized by their covalently closed loop structure that makes them more resistant to exonucleases, leading to greater stability and extended half-lives compared to linear mRNAs.¹ Another class of enhanced RNA therapeutics is saRNAs—an engineered class of linear mRNAs that replicate within host cells. They are typically derived from alphaviruses and include sequences encoding the RNA-dependent RNA polymerase (RdRp) that allows the replication of the saRNA, leading to the production of multiple copies of the RNA and, consequently, increased protein expression. Their safety and efficacy have been proved during COVID-19 pandemic where different saRNAs were approved for emergency use.²

The UTRs play critical roles in regulating translation efficiency and stability of both linear conventional circRNAs and saRNAs. RNA binding proteins (RBPs) play a pivotal role in the post-transcriptional regulation of mRNAs expression by binding to UTRs and influencing stability, translation, and subcellular localization. One key subset of RBPs is AU-rich element (ARE)-binding proteins, which regulate mRNA decay and translation. It has been estimated that 5%–20% of mammalian protein coding genes encode for AU-rich mRNA, making these elements a major cis-regulatory element into 3′UTR of mRNAs. In mammalian cells, AREs were discovered due to their role in directing host mRNAs for rapid degradation. However, more recently, it has been clarified that depending on the precise AU-rich sequence, position into the UTR, cellular context, subcellular localization, and specific stimulus, an ARE can interact with different ARE-binding proteins and contribute to either mRNA stabilization or degradation.³ The principal actors that regulate degradation of AU-rich elements are tristetraprolin, KH-type splicing regulatory protein, and AUF1 that binds to AREs and promotes mRNA decay by recruiting components of the deadenylation machinery or exonucleases. Contrariwise, HuR (ELAV-like protein 1) stabilizes mRNAs by preventing degradation and enhancing translation. It achieves this by competing with decay-promoting proteins and interacting with translation initiation factors. Peng et al. in 1998 provided evidence that HuR prevents the degradation of mRNAs by delaying their decay without affecting deadenylation.⁴ Later in 2019, Ripin et al. investigated the structure and function of the HuR protein in the context of AU-rich element-containing mRNAs. The study finds that in such cases, RNA recognition motif 3 of HuR counteracts the stabilizing function of the other HuR domains, adding complexity to HuR’s role in mRNA regulation.⁵ In February 2025, Ma and colleagues applied these basic research findings to the biotechnological application in in vitro transcribed RNA, where they explored an innovative strategy to transfer ARE into 3′UTRs to improve mRNA stability and consequently translation efficiency (Figure 1).⁶ The authors demonstrated that inserting AU-rich elements in the 3′UTR does not improve translation in terms of ribosome engaging, instead, AU-rich elements significantly enhance mRNA stability allowing to prolong mRNA half-life, leading to sustained protein expression. In support of this hypothesis, the translation efficiency of ARE-containing mRNA versus non-ARE mRNA appears to be very similar at extremely early observation time points. However, the insertion of ARE in the 3′ UTR ensures prolonged RNA half-life both in vitro and in vivo, thereby promoting sustained translation over several days. One of the significant findings of the study is that AU-rich elements positioned at the beginning of the 3′UTR result in the highest enhancement of translation efficiency. This suggests that the spatial arrangement of regulatory elements within the UTR can influence their interaction with RNA-binding proteins, ultimately affecting mRNA stability and translation (Figure 1). The authors reinforced the translational potential of their findings using cell lines of different origins and in vivo models. Also, by demonstrating that AREs enhance protein expression across various model proteins, the study establishes the generalizability of the approach for different therapeutic applications. In agreement with the speculative HuR-dependent mechanism of action, mRNA stability and reporter gene translation were significantly reduced in cells interfered for HuR. The immunoprecipitation experiment validated the interaction between AU-rich elements and HuR. Also, the authors systematically tested AU-rich elements from different genes that were also progressively shortened to identify in AUUUA the minimal domain required for functional interaction with HuR producing a translation enhancement of 3- to 5-fold.

Figure 1.

Iconographic description of AU-rich element (ARE) mechanism of action

ARE in certain configurations recruit HuR protein to 3′UTR, presumably preventing in vitro transcribed mRNA degradation. Graph depicts the concept of area under the curve, where stabilization of mRNA allows long-lasting mRNA translation and therapeutic effect.

The major takeaway from this study is the rational design of AREs to enhance translation efficiency. By systematically evaluating different ARE sequences, the researchers identified optimal configurations that enhance RNA stability and increase protein output. This optimization approach contrasts with conventional high-throughput screening methods, which can be resource-intensive. The authors validated the universality of their finding across different proteins and species and incorporating ARE into different UTRs demonstrating the broad applicability of this approach.

Despite the promising applications, there remain challenges in harnessing AREs for therapeutic purposes. First, to date, the optimal cell target to elicit a potent immune response to genetic vaccines is not fully understood. While the study focuses on HuR, the impact of the cellular context on ARE function needs further investigation. Indeed, mRNA stability and translation efficiency can vary across different cell types and physiological conditions where many different ARE-binding proteins can potentially recognize the same consensus and inducing opposite effects (i.e., stabilization vs. destabilization). For this reason, the specificity of RBP interactions with AREs must be carefully studied to ensure that introduced sequences do not inadvertently recruit destabilizing proteins. Also, minimal differences in ARE have been observed to dramatically affect mRNA stability and translation. The more emblematic case is the difference between ARE-V7 (5' UAUUUAUUUA 3') and ARE-V8 (5' AUUUAUUUA 3') where, while ARE-V8 increase mRNA translation compared to non-ARE construct, the presence of a single additional U in ARE-V7 completely abrogates mRNA translation. This finding underlines the need of refining sequence designs to maximize efficacy while minimizing unintended regulatory effects.

Finally, the impact of uridine replacement with modified nucleotides (e.g., N1-methyl-pseudouridine, m1ψ) on ARE structure should be carefully considered and addressed. This is particularly important for translating these genetic elements into different classes of RNA therapeutics that do not rely on classical modifications, such as circRNA and saRNA. Also in the case of viral vectors, enhancing the translation of the encoded gene by ARE could lead to more potent and long-lasting therapeutic effects (i.e., antigen, gene replacement, and payload).^7,8,9 However, in the context of gene transfer technologies that require nuclear transcription, the impact of ARE on nuclear export should be considered and examined.

Acknowledgments

This work was supported by POR Campania: piattaforma per lo sviluppo di nuove tecnologie vaccinali, PNRR CN3 National Center for Gene Therapy and Drugs based on RNA Technology, and PNRR PE13 One Health Basic and Translational Research Actions Addressing Unmet Needs on Emerging Infectious Diseases.

Declaration of interests

The authors declare no competing interests.