Cytoplasmic regulation of the poly(A) tail length as a potential therapeutic target

Abstract

Virtually all mRNAs acquire a poly(A) tail cotranscriptionally, but its length is dynamically regulated in the cytoplasm in a transcript-specific manner. The length of the poly(A) tail plays a crucial role in determining mRNA translation, stability, and localization. This dynamic regulation of poly(A) tail length is widely used to create posttranscriptional gene expression programs, allowing for precise temporal and spatial control. Dysregulation of poly(A) tail length has been linked to various diseases, including cancers, inflammatory and cardiovascular disorders, and neurological syndromes. Cytoplasmic poly(A) tail length is maintained by a dynamic equilibrium between cis-acting elements and cognate factors that promote deadenylation or polyadenylation, enabling rapid gene expression reprogramming in response to internal and external cellular cues. While cytoplasmic deadenylation and its pathophysiological implications have been extensively studied, cytoplasmic polyadenylation and its therapeutic potential remain less explored. This review discusses the distribution, regulation, and mechanisms of cytoplasmic polyadenylation element-binding proteins(CPEBs), highlighting their dual roles in either promoting or repressing gene expression depending on cellular context. We also explore their involvement in diseases such as tumor progression and metastasis, along with their potential as targets for novel therapeutic strategies.

INTRODUCTION

Poly(A) tail length plays a crucial role in determining mRNA fate, influencing translation, stability, and localization. Cells modulate the poly(A) tail dynamically to achieve precise temporal and spatial gene expression control, ensuring that protein synthesis occurs at the appropriate time and location to meet cellular and developmental requirements. This dynamic regulation occurs in both the nucleus and cytoplasm through the opposing actions of poly(A) polymerases (PAPs) and deadenylases, whose balance is crucial for maintaining cellular homeostasis. Disruptions in these processes are linked to various diseases, including cancers and neurological disorders (Liu et al. 2023).

Poly(A) tail length is intricately tied to multiple stages of the mRNA life cycle, including transcription termination, subcellular localization, mRNA quality control, translation efficiency, and mRNA decay, thus enabling precise regulation of gene expression. Generally, longer poly(A) tails enhance mRNA stability and increase translation efficiency, leading to upregulated gene expression, whereas shorter tails promote mRNA degradation (Eckmann et al. 2011; Weill et al. 2012; Passmore and Coller 2022; Biziaev et al. 2024).

Polyadenylation, typically occurring cotranscriptionally in the nucleus, involves the addition of nontemplated poly(A) tails up to 200–250 nt long to RNA polymerase II (Pol II)-synthesized RNAs, except replication-dependent histone transcripts. This process is mediated by polyadenylation signals (PASs) recognized by the cleavage and polyadenylation machinery, including more than 20 core proteins like the cleavage and polyadenylation specificity factors (CPSFs) and nuclear poly(A) polymerase (PAPα) (Tian and Manley 2017). Many mRNAs contain multiple PAS, leading to alternative cleavage and polyadenylation (APA) events that generate mRNA isoforms with varying 3′ untranslated regions (UTRs). The length of these UTRs influences the inclusion or exclusion of cis-acting elements, such as microRNA (miRNA)-binding sites or AU-rich elements (AREs), which recruit deadenylation complexes and regulate mRNA stability and translation (Mitschka and Mayr 2022).

As the poly(A) tail grows, it recruits nuclear poly(A)-binding proteins (PABPNs), which enhance PAP's binding and processivity, ultimately mediating the export of mRNA to the cytoplasm (Eliseeva et al. 2013). In the nucleus, the poly(A)-specific ribonuclease (PARN) functions as a deadenylase involved in quality control, shortening poly(A) tails as needed (Godwin et al. 2013). In the cytoplasm, PABPN1 is replaced by the cytosolic poly(A)-binding protein (PABPC), which protects mRNA from degradation and promotes translation. Cytoplasmic poly(A) tail length continues to be regulated dynamically by cytoplasmic PAPs, including atypical enzymes like GLD-2 and PAPD4, and by deadenylases. In mammals, deadenylation proceeds in a biphasic manner, primarily involving two conserved complexes: PAN2–PAN3 and CCR4-NOT (Weill et al. 2012; Liu et al. 2023). This cytoplasmic regulation allows for further fine-tuning of mRNA stability and translation efficiency after nuclear export. Enzymes can elongate or shorten the poly(A) tail based on cellular needs, modulating gene expression in response to various signals. This dual-layer regulation ensures that mRNA function is initially established in the nucleus and subsequently refined in the cytoplasm, providing precise control over protein synthesis to adapt to changing cellular conditions.

CYTOPLASMIC REGULATION OF POLY(A) TAIL LENGTH

Cis-acting elements, such as microRNAs (miRNAs) and AU-rich elements (AREs), primarily function to promote mRNA deadenylation and degradation. AREs were initially identified as conserved sequences composed of AUUUA pentamers located within the 3′ UTRs of specific mRNAs, particularly those encoding cytokines. These elements play a significant role in posttranscriptional regulation by influencing mRNA stability. It is estimated that ∼16% of human protein-coding genes contain AREs, many of which are associated with critical processes like inflammation, immune response, and cancer progression (Khabar 2010; Gruber et al. 2011; Beisang and Bohjanen 2012).

ARE-binding proteins (AUBPs) regulate mRNA stability by either promoting decay or stabilizing transcripts (Otsuka et al. 2019). For example, tristetraprolin (TTP) binds to ARE-containing mRNAs and facilitates degradation by recruiting the CCR4-NOT deadenylase complex, the exosome, and the Dcp1a/Dcp2 decapping complex, leading to translational repression (Lykke-Andersen and Wagner 2005; Bulbrook et al. 2018). The activity of TTP and similar AUBPs is often modulated by phosphorylation through signaling pathways such as p38/MAPK. Phosphorylation of TTP decreases its affinity for AREs, reducing its ability to target mRNAs for degradation (Tiedje et al. 2012). Conversely, other AUBPs, like HuR, stabilize mRNA transcripts by preventing their degradation (Brennan and Steitz 2001). This regulatory balance is critical for cellular homeostasis and has implications for diseases such as cancer, cardiovascular disorders, and neurodevelopmental diseases (Liu et al. 2023; Podszywalow-Bartnicka and Neugebauer 2024).

CPEB PROTEINS: STRUCTURAL AND FUNCTIONAL DIVERSITY

While numerous RNA-binding proteins (RNABPs) are involved in either promoting mRNA degradation or stabilization, only a select few have the ability to reactivate mRNAs that have been stored in an inactive state with shortened poly(A) tails. Among these, the cytoplasmic polyadenylation element-binding (CPEB) proteins are the most extensively characterized, and possibly the only RNABPs known to perform this function. CPEBs recognize and bind to cytoplasmic polyadenylation elements (CPEs) located in the 3′ UTRs of ∼20% of vertebrate genes, modulating mRNA translation through posttranscriptional regulation (Fig. 1; Pique et al. 2008; Duran-Arque et al. 2022).

FIGURE 1.

. Schematic representation of a CPEB bound to the CPE in the 3′ UTR of the target mRNA. The structure of the two RRMs and the ZZ-box is adapted with permission from Afroz et al. (2014). The intrinsically disordered N-terminal half of CPEBs is indicated, along with the 5′ and 3′ UTRs of the mRNA, the open reading frame (ORF), and the poly(A) tail.

All CPEB proteins possess a conserved C-terminal domain containing two RNA recognition motifs (RRMs), which are crucial for mRNA binding, while their N-terminal domains (NTDs) are variable (Fig. 2). This N-terminal region is responsible for their regulatory functions and interactions with other cellular factors, allowing CPEBs to adapt to diverse cellular environments and signaling cues (Fernandez-Miranda and Mendez 2012; Afroz et al. 2014). This structural versatility enables CPEBs to act as key regulators of mRNA stability and translation, thereby influencing numerous cellular processes.

FIGURE 2.

CPEB domains and regulatory phosphorylations. (Left) All four CPEBs share a similar structure, with a highly ordered C-terminal domain comprising two RRMs in tandem and a ZZ-box responsible for binding to their targets, and a highly variable NTD, which contains regulatory phosphorylation sites (activating sites in green, repressing sites in red; kinases indicated). CPEBs can also undergo alternative splicing, generating isoforms with potentially different behaviors and functions. (Right) CPEBs 2–4 are predicted to be intrinsically disordered and contain low-complexity regions (LCRs) within their variable NTDs. POND-score profiles of CPEBs 1–4 show stretches of predicted disordered regions.

CPEBs are nucleocytoplasmic shuttling proteins that regulate mRNAs at multiple levels (Fig. 3). In the nucleus, CPEBs participate in the definition of alternative PAS, and therefore alternative 3′ UTRs (Bava et al. 2013). CPEB can also recruit the nuclear deadenylase PARN (Kim and Richter 2006). In the cytoplasm, CPEBs regulate mRNA translation, either by assembling repressor complexes, in condensates with liquid-like properties (Fig. 4) (LLDs, which include the CCR4-NOT deadenylase that maintains target transcripts translationally silenced or by promoting cytoplasmic polyadenylation and subsequent translation through the recruitment of the atypical PAPs GLD-2 or GLD-4 [Igea and Mendez 2010; Guillen-Boixet et al. 2016; Duran-Arque et al.]). Coordinated with the temporal regulation of translation, the CPEB-assembled translational repression complexes can be localized and subsequently locally activated to generate spatial regulation of gene expression (Huang and Richter 2004; Nagaoka et al. 2012; Pascual et al. 2020b; Huang et al. 2023).

FIGURE 3.

Proposed model of nucleocytoplasmic functions of CPEB1. In the nucleus, CPEB1 facilitates the recruitment of CPSF to suboptimal (upstream) polyadenylation sites (PAS), thereby promoting the generation of shorter 3′ UTRs. In the cytoplasm, nonphosphorylated CPEB1 assembles a repression complex by recruiting deadenylases and associated cofactors, effectively blocking the mRNA cap and rendering the mRNA translationally silent. These mRNPs (messenger RNP particles) can associate with motor proteins, enabling their subcellular localization along microtubules. Upon phosphorylation, CPEB1 undergoes activation, leading to the remodeling of the mRNP complex. This remodeling facilitates the recruitment of CPSF and the cytoplasmic PAPs Gld2 or Gld4, which extend the poly(A) tail. Simultaneously, CPEB1 recruits cap-ribose methyltransferases, promoting the formation of CapI and CapII structures, thereby enhancing mRNA stability and translational efficiency.

FIGURE 4.

The activity of CPEBs 2–4 as translation regulators depends on phase separation. Unphosphorylated CPEBs undergo liquid–liquid phase separation, repressing the translation of target mRNAs. Hyperphosphorylation by ERK2 and Cdk1 induces changes in the charge pattern, leading to condensate disassembly and promoting translational activation of CPEB4 targets. Phase separation properties rely on the intrinsically disordered NTD. Image adapted with permission from Guillen-Boixet et al. (2016).

The CPEB family consists of four members in vertebrates, classified into two subfamilies: CPEB1 and CPEB2–4. These two groups have differential properties in target/motif recognition, formation and dynamics of large-order complexes and regulation by posttranslational modifications. CPEB1 recognizes canonical CPEs (UUUUA(1-2)U), whereas CPEB2–4 recognize both canonical and G-variant CPEs (UUUUGU). This differential binding implies that, while CPEB1 targets are shared with CPEB2–4, this second CPEB subfamily has also other specific targets. CPEB1 assembles into large ribonucleoprotein (RNP) complexes that exhibit structural stability and associate with the cytoskeleton for mRNA localization, aligning with its interaction with dynein and kinesin (Fig. 3; Huang et al. 2003). On the other hand, condensates assembled by CPEB2–4 showed properties of canonical LLDs in terms of size, morphology, and dynamic properties (Fig. 4).

Regulation by phosphorylation also defines two subfamilies (Fig. 2). Thus, CPEB1 is activated by a single phosphorylation event mediated by Aurore kinase A (AurkA), leading to the remodeling of its mRNP complexes but not their inclusion into LLDs (Duran-Arque et al. 2022). Subsequent multiple proline-directed phosphorylation events promote CPEB1 degradation. On the other hand, CPEB2–4 are activated through multiple proline-directed phosphorylation events (by ERK and Cdk1), which promote the dissolution of LLDs (Duran-Arque et al. 2022). Therefore, the two CPEB subfamilies are regulated through distinct signaling pathways and mechanisms and more importantly, both subfamilies cannot be activated simultaneously. Indeed, AurkA is specifically activated at pro-metaphase and upon synaptic stimulation, while CDK1/ERK are activated starting from anaphase during cell cycle, and ERK as part of the stress responses. This implies that, for cells that coexpress the two subfamilies as well as for mRNAs with canonical CPEs, there will be a competitive equilibrium between a CPEB acting as a repressor and other CPEBs acting as activators. mRNAs with noncanonical G/CPEs would not be subjected to these opposing actions. In addition, regulation by multiple phosphorylation sites in CPEB2–4, compared to a single site in CPEB1, enables an ultrasensitive response in the CPEB2–4 subfamily. Multiple cooperative phosphorylation events enhance the sensitivity of successive steps, resulting in a steep, sigmoidal output. Additionally, requiring phosphorylation of only a subset of sites for activation balances low- and high-end thresholds, further improving system responsiveness. (Ferrell and Ha 2014).

Changes in CPEB expression, particularly in epithelial cells, are linked to tumor growth and vascularization, highlighting their involvement in cancer biology (Ortiz-Zapater et al. 2011; Fernandez-Miranda and Mendez 2012; D'Ambrogio et al. 2013; Calderone et al. 2016). The properties of the regulation of poly(A) tail length by CPEBs generate a mechanism to define temporal and spatial windows of gene expression that can be rapidly reprogrammed in response to intracellular and extracellular cues. Accordingly, this dynamic regulation of the poly(A) tail length is extensively utilized to create posttranscriptional gene expression programs where precise temporal or spatial patterns are required.

Therefore, both ARE- and CPE-dependent mechanisms generate a dual regulatory equilibrium in which the poly(A) tail length is dynamically modulated by opposing cis-acting elements, CPEs and AREs, which in turn recruit trans-acting factors competing for opposite outcomes (CPEB1/CPEB2–4 or TTP/HuR) (Fig. 5). CPEB proteins and TTP are both RNABPs that regulate mRNA stability and translation, but they generally exert opposing effects. CPEB and TTP have opposing functions in various cellular contexts. CPEB generally acts to stabilize mRNAs and promote their translation, enhancing protein expression, whereas TTP promotes mRNA degradation and decreases protein expression. Their interplay is critical for fine-tuning gene expression in response to cellular and environmental cues, balancing processes such as inflammation, cell cycle progression, synaptic plasticity, and tumor growth.

FIGURE 5.

Cytoplasmic regulation of poly(A) tail length is controlled by a double equilibrium of cis-acting elements (AREs vs. CPEs) and trans-acting factors (TTP vs. HuR and CPEB1 vs. CPEB4). The activity of the trans-acting factors is, in turn, regulated by kinases acting in a coordinated manner on AREBP and CPEBs.

These trans-acting factors are themselves regulated, in a coordinated manner, by signal transduction pathways that are, in turn, translationally controlled by CPEs and AREs, establishing self-sustained, through interlinked positive and negative feedback loops, gene expression programs (Belloc and Mendez 2008; Suñer et al. 2022). This coordination ensures that cellular responses are tightly regulated and adaptive, allowing for precise modulation of gene expression in response to external stimuli and maintaining cellular homeostasis.

CPEB PROTEINS IN DISEASE CONTEXTS

Neurological disorders

Local protein synthesis at synapses is critical for synaptic plasticity, the cellular basis of learning and memory. Newly synthesized proteins are believed to “tag” activated synapses, distinguishing them from unstimulated ones and thereby establishing a form of cellular memory. This process requires the transport of mRNA from the soma to dendrites, coupled with translational activation at synapses in response to neuronal stimulation. CPEBs are essential for synaptic plasticity and memory, playing key roles in regulating mRNA localization, translation, and stability, which are vital for neuronal function. Dysregulation of CPEB expression or activity has been implicated in cognitive impairments, neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), epilepsy, Huntington's disease, Alzheimer's disease, and fragile X syndrome (FXS) (Pico et al. 2021; Huang et al. 2023).

Notably, different CPEB family members can contribute to the same phenotype by participating in distinct sequential steps. For instance, CPEB1 interacts with fragile X mental retardation protein (FMRP), the product of the FMR1 gene associated with FXS, a neurodevelopmental disorder on the autism spectrum (ASD) and the most common inherited cause of intellectual disability. Interestingly, depletion of CPEB1 has been shown to compensate for mutant FMRP, presumably due to its role in mRNA translational repression and localization (Udagawa et al. 2013). CPEB4 also plays a significant role in ASD. Increased skipping of the neuron-specific exon 4 in CPEB4 (nCPEB4Δ4) has been observed in idiopathic ASD. This splicing alteration correlates with reduced poly(A) tail length and diminished expression of several ASD-risk genes. Transgenic expression of nCPEB4Δ4 in mouse brains induced autistic-like behaviors, including changes in anxiety, vocalization, and social interaction. However, CPEB4 haploinsufficiency or loss does not phenocopy nCPEB4Δ4, suggesting that the mutant variant acts as a dominant negative, disrupting the function of all CPEB proteins (Parras et al. 2018). Our findings also reveal that maintaining a precise ratio of microexon 4 inclusion is essential for preserving the dynamic state of neuronal CPEB4 (Garcia-Cabau et al. 2024). Neuronal CPEB4 forms condensates that dissolve upon depolarization, transitioning from translational repression to activation. This phase transition is mediated by heterotypic interactions between the microexon and a cluster of histidine residues. These interactions kinetically stabilize the condensates by competing with homotypic interactions that otherwise promote irreversible aggregation. The phase equilibrium of neuronal CPEB4 is regulated by changes in the protonation state of histidine residues in the intrinsically disordered NTD in response to local pH fluctuations during depolarization. These changes modulate intermolecular interactions, promoting reversible condensation. Thus, microexon 4 plays a crucial role in enhancing both the thermodynamic and kinetic stability of repressive CPEB4 condensates, effectively preventing aggregation. Importantly, even slight variations in microexon 4 inclusion significantly alter aggregation tendencies. In ASD, the observed reduction in microexon 4 inclusion levels leads neuronal CPEB4 to adopt a more solid-like state sequestering CPE-containing mRNAs and other CPEBs present in the same aggregates. Intriguingly, exogenous addition of a peptide containing the microexon can restore the dynamic properties of condensates formed by nCPEB4Δ4 in a dose-dependent manner. This finding suggests potential therapeutic strategies for targeting the pathological effects of microexon 4 skipping in ASD.

Cancer

CPEBs are integral to cell cycle regulation, influencing tumor development and progression by modulating mRNA translation through poly(A) tail length dynamics. During meiotic and mitotic cell cycle transitions, changes in poly(A) tail length facilitate the translation or degradation of mRNAs encoding critical proteins like cyclins and cyclin-dependent kinases (CDKs). Generally, phosphorylated CPEBs, in conjunction with HuR, promote cell cycle progression, whereas unphosphorylated CPEB1 and TTP act as inhibitors, repressing or destabilizing their target mRNAs to halt cell cycle progression (Villalba et al. 2011; Ivshina et al. 2014; Galloway et al. 2016; Pascual et al. 2020b; Haga et al. 2024).

Both CPEBs and AREBPs are implicated in tumor suppression and cancer progression. For example, TTP and HuR, which either destabilize or stabilize prosurvival mRNAs, have been widely studied in cancer biology (Ross et al. 2012; Finan et al. 2023). Cytoplasmic HuR plays a key role in stabilizing and enhancing the translation of prosurvival mRNAs involved in cellular stress responses, including those triggered by hypoxia, radiotherapy, and chemotherapy. Its ability to bind and stabilize these mRNAs enhances the cell's capacity to survive and adapt under stress conditions, which is particularly advantageous for cancer cells. HuR's overexpression and elevated activity in cancer cells, compared to its relatively lower levels in normal cells, make it a promising target for cancer therapy. Targeting HuR could disrupt the survival mechanisms that cancer cells rely on, potentially sensitizing them to treatments and reducing tumor progression (see below).

CPEBs proteins play diverse and context-dependent roles in cancer progression by regulating mRNAs that control cell growth, survival, and tumor suppression (Fernandez-Miranda and Mendez 2012; D'Ambrogio et al. 2013). Depending on the cellular context and specific CPEB isoform involved, they can either promote oncogenesis or act as tumor suppressors. Although their functions in cancer have not yet been systematically analyzed, and there are relatively few cases with established causality, CPEBs have been shown to influence key processes such as cell cycle regulation, cell death, and angiogenesis. This variability highlights the complexity of their roles and suggests that CPEBs could be both potential biomarkers and therapeutic targets, depending on the type and stage of cancer. CPEB1, in particular, is generally associated with tumor suppression, while CPEB4 and CPEB2 are more often linked to malignancy. Studies show that CPEB1, CPEB2, and CPEB4 are implicated in breast cancer, where they either enhance tumor growth or facilitate metastasis (Nagaoka et al. 2016; Lu et al. 2017; Pascual et al. 2020a). Additionally, CPEB1 and CPEB3 are frequently downregulated in ovarian cancer (Hansen et al. 2009), correlating with the aggressive status of the disease. Both CPEB1 and CPEB4 are involved in glioblastoma development (Ortiz-Zapater et al. 2011; Boustani et al. 2016; Galardi et al. 2016; Hui et al. 2018). In hepatocellular carcinoma, CPEB4 plays a pivotal role in regulating ferroptosis and iron homeostasis, key processes that influence tumor progression (Delgado et al. 2024). Low CPEB4 expression correlates with poor prognosis, reduced ferroptotic sensitivity, and increased tumor aggressiveness in both human and mouse models. Mechanistically, CPEB4 enhances the translation of hepcidin, a key regulator of intracellular iron levels under stress conditions. Its depletion results in reduced hepcidin, elevated ferroportin, lower intracellular iron, and impaired lipid peroxidation, thereby diminishing ferroptotic cell death and providing liver cancer cells a survival advantage (Delgado et al. 2024). CPEB4 also exhibits a biphasic expression pattern, with high levels during early hepatocellular carcinoma transitioning to lower levels in advanced stages (Tsai et al. 2016), highlighting its stage-specific tumor-suppressive functions. These findings suggest that therapeutic strategies aimed at activating CPEB4 or inhibiting its antagonist, CPEB1, hold promise for mitigating liver cancer progression. Furthermore, CPEB4 contributes to gastric cancer progression by promoting growth and metastasis via ZEB1-mediated epithelial-mesenchymal transition (EMT), illustrating its critical role in malignancy enhancement (Cao et al. 2018). CPEB4 also indirectly influences colorectal cancer development through inflammation resolution (Sibilio et al. 2022). In contrast, CPEB3 acts as a tumor suppressor in gastric cancer (Chen et al. 2022), emphasizing the diverse roles of CPEB proteins across different cancers. In pancreatic ductal adenocarcinoma (PDA) and glioblastoma, aberrant CPEB4 expression promotes metastasis by activating tissue plasminogen activator (tPA) mRNA, which is usually silenced in homeostatic conditions (Ortiz-Zapater et al. 2011). CPEB4 also contributes to melanoma progression by regulating the translation of mRNAs linked to cell migration and invasion (Perez-Guijarro et al. 2016). In contrast, CPEB1 generally acts as an antitumoral agent in various cancers. For example, it has been shown to suppress tumor growth in pancreatic cancer, gastric cancer, and melanoma (Shoshan et al. 2015; Zhang et al. 2024). This complex interplay between CPEB family members underscores their context-specific functions in cancer biology. While CPEB1 often functions as a tumor suppressor, CPEB4 frequently supports tumorigenic processes, highlighting the need for precise therapeutic targeting based on the specific cancer type and CPEB protein involved.

Immune regulation and inflammation

CPEBs are emerging as important regulators in immune responses and inflammation. Poly(A) tail dynamics are key in creating temporal windows for gene expression, which are essential for controlling the tumor inflammatory immune environment. RBPs, such as CPEBs, interact with cis-RNA elements to regulate mRNA stability and translation, thus influencing immune cell function and metabolism (Turner and Diaz-Muñoz 2018).

A well-studied RBP-mediated mechanism related to inflammation involves the p38 MAPK-regulated balance between TTP (which destabilizes mRNAs) and HuR (which stabilizes them), controlling the timing and levels of proinflammatory cytokines (Suñer et al. 2022). Disruption in this balance can result in either reduced cytokine production or chronic inflammation, leading to autoimmune diseases or impaired antitumor immune responses (Salerno et al. 2018a,b).

Although much less studied, recent data also suggest a role for CPEBs in tumor-associated immune responses. For instance, CPEB4-transduced dendritic cells have been shown to enhance the number of IFN-γ secreting cytotoxic T cells in gliomas, reducing tumor growth and increasing life span in mouse models (Peng et al. 2014). Lipopolysaccharide (LPS)-injected CPEB1 knockout mice produce significantly elevated levels of IL-6 and other proinflammatory cytokines, exhibiting enhanced sensitivity to endotoxic shock. These findings indicate that CPEB1 plays a protective role in regulating the innate immune response (Ivshina et al. 2015). Additionally, CPEB4 mediates a novel adaptive mechanism of the unfolded protein response (UPR), aimed to resolve the ER-stress caused by the accumulation of unprocessed proteins in the ER lumen, essential for the function of CD8⁺ T cells. Depletion of CPEB4 impaired T-cell-mediated antitumor responses, underscoring its role in sustaining T-cell effector functions by mitigating chronic ER stress generated by the secretory phenotype of effector T cells (Fernandez-Alfara et al. 2023). In macrophages, CPEB4 stabilizes anti-inflammatory mRNAs during inflammatory responses, promoting the resolution of inflammation. Mice lacking CPEB4 show increased inflammatory responses and impaired survival, indicating that CPEB4 is essential for maintaining macrophage function and preventing excessive inflammation (Suñer et al. 2022). CPEB4's role extends to responses to LPS stimulation, where its absence led to elevated inflammatory signaling and reduced anti-inflammatory cytokines (Suñer et al. 2022). Furthermore, CPEB4's involvement in obesity-related inflammation has also been documented. CPEB4 drives posttranscriptional reprogramming in adipocytes under obesity conditions, affecting macrophage polarization and migration. CPEB4 regulates proinflammatory factors such as CCL2 and TLR4, linking its activity to inflammation modulation in adipose tissue and broader immune system dynamics (Pell et al. 2021). CPEB4-deficient mice showed exacerbated inflammation and altered immune responses, including decreased IL-22 expression, which influenced both acute inflammation resolution and tumor progression (Sibilio et al. 2022).

Overall, these findings emphasize the critical role of the CPEB1/CPEB4 balance in immune regulation, inflammation resolution, and tumor dynamics. Its involvement in stress adaptation, macrophage function, and tumor immunity highlights its potential as a therapeutic target for modulating immune responses and addressing inflammatory and cancerous conditions.

Chronic stress responses

Metabolic stress, or a highly secretory phenotype, often triggers endoplasmic reticulum (ER) stress due to the accumulation of unfolded or misfolded proteins, leading to activation of the UPR. The UPR consists of three signaling pathways mediated by activating transcription factor-6 (ATF6), inositol-requiring enzyme-1α (IRE-1α), and the eIF2α kinase PERK. Phosphorylation of eIF2α reduces overall protein synthesis, while simultaneously allowing the selective translation of specific mRNAs containing short upstream open reading frames (uORFs). These initial stress responses either facilitate the resolution of stress or, if the stress cannot be resolved, promote apoptosis by inducing the transcription factor CHOP (Harding et al. 2000; Wek et al. 2006; Wang and Kaufman 2014; Hetz et al. 2020). The primary goal of these early responses is to manage acute, temporary stress or trigger programmed cell death if necessary.

However, in cases of chronic stress, such as prolonged exposure of hepatocytes to a high-fat diet (HFD), the ER stress is persistent but less severe, and it does not lead to apoptosis. This chronic ER stress response is partially regulated by CPEB4, which activates the translation of mRNAs containing CPEs (Fig. 6). The proteins produced by these mRNAs help alleviate metabolic ER stress without causing a general shutdown of protein synthesis. Unlike the response triggered by eIF2α phosphorylation, the CPEB4-mediated response can be sustained over long periods without harmful effects on the cell (Maillo et al. 2017). The translation of Cpeb4 mRNA itself is initially activated by uORFs in its 5′ UTR (Maillo et al. 2017) and is later maintained by CPE elements in its 3′ UTR through a positive feedback loop (Igea and Mendez 2010).

FIGURE 6.

Model of the adaptive response to chronic stress. Sustained UPR occurs in two sequential phases. Acute phase: transient activation via eIF2α phosphorylation, allowing the permissive translation of uORF-containing mRNAs, including CPEB4 mRNA. Chronic phase: mediated by translational activation and increased stability of CPE-containing transcripts, including CPEB4.

CPEB4, unlike other members of the CPEB family, is encoded by a circadian-regulated and uORF-containing mRNA that responds to acute stress and eIF2α phosphorylation. The levels of CPEB4 mRNA fluctuate in a circadian rhythm, allowing the protein to prepare cells for periods of increased ER stress (Balvey and Fernandez 2021). This function of CPEB4 plays a critical role in the adaptation to chronic ER stress, particularly in highly secretory immune cells, where it regulates cytokine production. CPEB4 allows effector CD8⁺ T cells to maintain their high anabolic and secretory capacity and thereby supports their antitumor activity (Fernandez-Alfara et al. 2023). Similarly, CPEB4 is essential for other cellular adaptations associated with chronic activation of the integrated stress response, such as in cardiac hypertrophy and heart failure, where it regulates genes involved in cardiac function and stress response (Riechert et al. 2021). Additionally, CPEB1 and CPEB4 work together to regulate the translation of VEGF mRNA, promoting angiogenesis in chronic liver disease (Calderone et al. 2016).

Liver and metabolic disorders

Although the involvement of multiple CPEB proteins in metabolic disorders is not extensively documented, CPEB1 and CPEB4 have been linked to critical metabolic processes. For example, CPEB1 plays a role in regulating insulin signaling pathways (Alexandrov et al. 2012), while CPEB4 influences lipid metabolism and glucose homeostasis (Burns and Richter 2008; Maillo et al. 2017). Notably, CPEB4 is required for adipose tissue expansion and inflammation under obesity conditions. It also modulates gut microbiota, shifting it toward a more pathogenic profile. Studies have shown that CPEB4 depletion in HFD models reduces adipogenesis and attenuates the proinflammatory characteristics of both visceral fat depots and the microbiome (Pell et al. 2021).

CPEB4's role in liver disease is complex and stage-dependent, acting as both a protector and a promoter of pathology. In the early stages of liver steatosis, particularly under HFD and obesity conditions, CPEB4 has a protective function. It supports mitochondrial fatty acid oxidation and regulates ER stress, helping to maintain metabolic balance (Maillo et al. 2017). Mice deficient in CPEB4 demonstrate lipid accumulation, hyperglycemia, and impaired stress responses, highlighting CPEB4's critical role in lipid and glucose homeostasis. The impairment of lipid metabolism due to CPEB4 deficiency indirectly disrupts glucose metabolism by inhibiting hepatic insulin signaling, which leads to hepatosteatosis and the accumulation of unfolded proteins (Maillo et al. 2017).

As liver disease progresses to advanced stages, CPEB4's role shifts from protective to detrimental. It becomes a contributor to liver fibrosis by activating hepatic stellate cells (HSCs) through the upregulation of PFKFB3, a key enzyme in glycolysis (Mejias et al. 2020). This shift toward glycolytic reprogramming fuels HSC proliferation and fibrogenic activity, resulting in excessive collagen deposition and compromised liver structure. Molecularly, CPEB4 drives fibrogenesis by binding to CPE sequences in PFKFB3 mRNA, thereby increasing its translation and promoting HSC activation. This process is a key factor in the excessive extracellular matrix (ECM) production characteristic of liver fibrosis. Furthermore, CPEB4 facilitates pathological angiogenesis in liver cirrhosis by enhancing the translation of vascular endothelial growth factor (VEGF) mRNA, leading to increased endothelial cell proliferation and new blood vessel formation (Calderone et al. 2016). Additionally, CPEB4 protects against liver cancer progression by regulating iron-dependent ferroptotic cell death in liver tumor cells (Delgado et al. 2024). CPEB4 deficiency reduces ferroptosis sensitivity, thereby promoting tumor progression.

The dual role of CPEB4 in liver disease highlights the need for a nuanced therapeutic approach. Targeted modulation of CPEB4 activity could potentially mitigate its harmful effects in fibrosis and pathological angiogenesis, while preserving its beneficial functions in managing ER stress and maintaining metabolic homeostasis. Such precision therapies could provide a promising strategy to address the multifaceted challenges associated with chronic liver disease.

POLYADENYLATION AND CPEBs AS THERAPEUTIC TARGETS

CPEBs, like many RNABPs, have traditionally been considered challenging drug targets due to their structural properties and the lack of well-defined small molecule binding sites. The conservation of canonical RNA-binding domains (RBDs) and the intrinsically disordered regions (IDRs) present in many RBPs add further complexity to developing small molecule inhibitors. Despite these challenges, recent advancements in high-throughput screening technologies for RBP-small molecule interactions are opening new avenues for therapeutically targeting these regulators of poly(A) tail length.

CPEBs are divided into two subfamilies, each exhibiting distinct, often opposing functions, integrating various cellular signals to modulate gene expression (Duran-Arque et al. 2022). The NTD of CPEBs is highly variable across the four isoforms, featuring IDRs that likely serve as docking sites for protein–protein interactions. For instance, CPEB1's NTD contains phosphorylation sites for Aurora A kinase, ERK/Cdk1, and Plk1, which regulate its activity through ubiquitination and subsequent degradation via the PEST box. In contrast, CPEB2–4 harbor similar regulatory phosphorylation sites controlled by ERK/p38 kinases and uniquely regulate the formation of liquid–liquid phase-separated assemblies that sequester inactive mRNAs (Guillen-Boixet et al. 2016; Duran-Arque et al. 2022).

All CPEB proteins can interact with the CCR4-NOT complex to repress translation and, upon phosphorylation, recruit the GLD2 complex to activate translation. Notably, higher-order complexes formed by CPEB1 resemble canonical RNP assemblies, requiring mRNA binding for their formation. In contrast, CPEB2–4 are capable of forming liquid–liquid phase-separated (LLD) droplets, which are characterized by their unique size, morphology, and dynamics, independent of RNA binding. This structural and functional divergence highlights the unique distribution of disordered regions within the NTDs of the two subfamilies. Upon coexpression, different CPEBs colocalize into larger aggregates while preserving their individual characteristics, indicating spatial heterogeneity within the LLDs. This probably reflects fusion events of smaller, and CPEB-specific, aggregates into larger ones. Thus, large aggregates contain subdomains for each of the CPEBs subjected to local regulation by their specific cofactors. At the same time, the phase transition dynamic properties of the large aggregates become unified, explaining the dominant negative effect of the CPEB4D4 splice variant explained above. Their proximomes (proteins into close spatial proximity identified by BioID, where a fusion between CPEBs and a mutant biotin ligase catalyzes the covalent attachment of biotin to nearby proteins within a range of 10 nm, labeling them for subsequent identification via mass spectrometry), comprise both common and CPEB-specific cofactors, with CPEB1 undergoing remodeling after a single phosphorylation event, while CPEB2–4 are modulated by multiple proline-directed phosphorylation events that govern their phase separation.

The RRMs in CPEB2–4 are highly conserved, showing 96% sequence identity, while CPEB1 shares only ∼46% identity with the other isoforms. The tandem arrangement of RRMs in CPEB1 and CPEB4 is unique, enabling both domains to bind a continuous RNA oligonucleotide (Afroz et al. 2014). Additionally, CPEB RRMs contain extensions to the canonical RRM fold; for example, CPEB1 has an extension that interacts between the region upstream of RRM1 and the helical face of RRM2, enhancing its specificity for canonical CPEs (UUUUA_(1-2)U). In contrast, CPEB2–4 can also bind “G-variants” (UUUUGU), expanding their RNA target specificity.

Integrating mechanistic insights with innovative screening methodologies holds significant potential for discovering novel compounds that target CPEBs. Recent assays targeting RBPs have emerged, such as AlphaScreen technology, which identifies small molecules that modulate cytoplasmic poly(A) tail length regulation (Julio and Backus 2021; Bertoldo et al. 2023). Understanding the dynamic equilibrium governing polyadenylation and deadenylation opens new therapeutic strategies to selectively enhance or inhibit these processes. In particular, the mechanistic understanding of CPEBs functions, structures, and regulation is being exploited in multiple directions:

Proximity-based biotinylation strategies, like Bio-ID labeling, are expanding the range of potential therapeutic targets within polyadenylation and deadenylation complexes. These methods are beginning to shed light on the complex compositions and dynamics associated with CPEB assemblies, enabling the identification of new molecular players and potential drug targets (Duran-Arque et al. 2022).

Targeting kinase pathways that regulate CPEBs also represents a promising approach. Both ARE and CPE elements are coregulated by proline-directed kinases like p38 and ERK, which activate CPEB4 and HuR, while inhibiting TTP and CPEB1 function (Duran-Arque et al. 2022; Suñer et al. 2022). Inhibitors targeting these kinases could therefore shift the balance toward deadenylation and inactivation of mRNAs associated with these elements, including those encoding proinflammatory cytokines and other critical proteins.

High-throughput screening technologies, such as proximity-based fluorescent assays (e.g., AlphaScreen), are proving effective in identifying small compounds that can disrupt RBP–RNA interactions. The distinctive RNA recognition mechanisms employed by CPEB RRMs may enhance selectivity for drug candidates. The structural differences among CPEB RRMs, alongside the specific binding patterns of AREBPs and CPE-binding proteins (CPEBPs), suggest the feasibility of identifying compounds that can selectively tilt the balance toward polyadenylation or deadenylation. For instance, HuR, a prominent AREBP known for stabilizing mRNAs involved in cancer, inflammation, and stress responses, has several small-molecule inhibitors in development that reduce levels of HuR-regulated mRNAs in an ARE-dependent manner. These include MS-444 (blocks HuR dimerization), KH-3 (prevents HuR from binding to AREs), deltonin (downregulates HuR expression), dehydromutactin (disrupts HuR–mRNA interactions), and azaphilone derivatives (inhibit HuR's function) (Bertoldo et al. 2023; Li and Kang 2023).

Beyond small molecules, RNA-PROTACs offer a novel approach. These compounds utilize small RNA mimetics to target the RNA-binding site of RBPs, facilitating their proteasomal degradation via a conjugated E3-recruiting peptide (Ghidini et al. 2021). The specificity of cis-acting element recognition for RBPs controlling cytoplasmic poly(A) tail length allows for targeted interventions against the ARE-BPs TTP and HuR (AUUUA), CPEB1–4 (UUUUAU), or CPEB2–4 (UUUUGU), potentially correcting dysregulated gene expression associated with diseases.

Exploiting the gene expression reprogramming linked to imbalanced CPEB expression in various pathological conditions offers promising avenues for therapeutic intervention. Targeting CPEBs may allow for the ectopic expression or suppression of factors that influence cellular function, survival, or apoptosis, depending on the disease context. For instance, PDA cells exhibit a dependency on elevated levels of CPEB4 compared to normal tissue (Ortiz-Zapater et al. 2011). This dependency can be leveraged therapeutically by engineering oncolytic adenoviruses with CPE regulatory sequences in the 3′ UTR of the E1A gene. Such viruses achieve selective efficacy in targeting cancer cells (oncoselectivity) while minimizing off-target effects on normal cells (Villanueva et al. 2017). This approach underscores the potential of CPEBs to direct tissue-specific therapies with reduced systemic toxicity.

The exploration of CPEBs and other RNABPs as therapeutic targets represents a complex but promising opportunity for drug development. Advancing our understanding of their mechanistic roles—especially how they regulate mRNA stability, translation, and poly(A) tail dynamics—can facilitate the identification of new intervention points. Innovative screening technologies, including high-throughput assays and proximity-based biotinylation strategies, are critical tools that can uncover selective compounds or RNA-based therapies targeting these RBPs. By combining mechanistic insights with these state-of-the-art screening methodologies, the therapeutic potential of CPEBs can be harnessed to address a variety of diseases, including cancers, inflammatory disorders, and neurodegenerative conditions.

CONCLUSION

CPEBs appear to have evolved exclusively in multicellular organisms, likely associated with meiotic cell divisions (Fernandez-Miranda and Mendez 2012). The initial CPEB was likely CPEB1 (Orb in Drosophila), with the other subfamily members arising through gene duplication (CPEB2–4 in vertebrates, Orb2 in Drosophila). The first duplication event into two subfamilies allowed for an expansion of mRNA targets and the diversification of regulation through different kinases and phase transitions (Duran Arque et al. 2022). The subsequent duplication, from Orb2 to CPEB2–4, had a more subtle impact. Although CPEB2–4 proteins appear to have distinct functions and tissue-specific expression patterns, they are largely functionally interchangeable, suggesting that their specialization is more related to their tissue-specific expression than to their individual molecular functions. Throughout evolution, CPEBs seem to have developed roles that help overcome the physiological constraints of transcriptional regulation. The original function of CPEBs likely involved the regulation of maternal mRNAs during meiosis, occurring when chromatin is fully condensed, and transcription is inhibited. A similar scenario arises during the mitotic M phase (Pascual et al. 2020a,b). CPEB4, in particular, mediates the chronic stress response by preventing the transcription of new mRNAs under stress conditions, where transcription errors are more likely. This allows for the reactivation of preexisting, stored mRNAs. A prominent example of this is seen in synaptic translation in neurons, where CPEBs regulate the repression, localization, and local activation of mRNAs. This regulation holds significant potential for determining cellular polarity and functional compartmentalization, processes that cannot be achieved through transcriptional mechanisms alone. This specialization in particular physiological processes, in turn, determines the pathological scenarios where CPEBs have been implicated.

CPEB proteins play diverse and complex roles across various disease contexts, often exhibiting opposing functions that depend on the cellular environment and specific isoform involved. This functional dichotomy presents both challenges and opportunities for therapeutic intervention. For instance, while CPEB1 generally acts as a tumor suppressor by promoting apoptosis and inhibiting cell proliferation, CPEB4 often has the opposite effect, enhancing cell proliferation and inhibiting apoptosis, thereby supporting tumor growth and survival. These contrasting functions highlight the difficulty in developing targeted therapies, as the roles of CPEBs can vary dramatically depending on the context.

Despite these challenges, the intricate regulatory mechanisms of CPEBs present unique opportunities for therapeutic innovation. By precisely targeting the functional antagonisms within CPEB isoforms, it may be possible to develop effective treatments that exploit these dual roles to address specific pathological conditions. Understanding the balance between CPEB isoforms in different diseases could lead to the development of tailored therapeutic strategies, offering a pathway toward precision medicine.

Integrating transcriptomic and proteomic approaches is critical to identifying CPEB target genes and proteins and to unraveling their involvement in processes such as cell proliferation, stress response, and immune regulation. This comprehensive analysis will be instrumental in discovering novel biomarkers and therapeutic targets, paving the way for more precise and effective interventions.

Looking ahead, several key research directions need to be pursued to fully unlock the therapeutic potential of CPEB proteins. A deeper understanding of the context-dependent roles of CPEBs, such as the pro-oncogenic activity of CPEB4 in cancers and its involvement in liver diseases, is essential. Future studies should focus on elucidating the specific molecular mechanisms through which CPEBs influence disease progression and how they interact with other signaling pathways. Advanced in vivo models and emerging technologies will also be crucial for translating these insights into clinical applications.

Overall, a more refined understanding of CPEBs will be instrumental in developing targeted therapies for a range of conditions. By addressing these complexities, future research has the potential to harness the full therapeutic potential of CPEBs, contributing to the advancement of precision medicine.