Argonaute (AGO) proteins are central components of the microRNA (miRNA) pathway, and among the four AGO isoforms identified in humans, only AGO2 has intrinsic endonuclease activity (Nakanishi, 2022). Mature miRNAs are loaded onto AGO2, which forms the miRNA-induced silencing complex to mediate post-transcriptional gene silencing (Gebert and MacRae, 2019). The extent of complementarity between the miRNA and its target mRNA determines the silencing mechanism. Complete complementarity triggers AGO2-mediated cleavage of the target mRNA, whereas partial complementarity typically results in miRISC-mediated translational repression, deadenylation and decapping (Shang et al., 2023). In stem cells, AGO2 has emerged as a regulator of pluripotency (Müller et al., 2020). Notably, knocking out AGO2 in mouse embryonic stem cells (mESCs) increases self-renewal and decreases differentiation (Liu et al., 2024). AGO2 associates with the pro-differentiation miRNA let-7. Elevated AGO2 levels increase let-7 expression and disrupt pluripotency in mESCs (Liu and Chen, 2021). Additionally, AGO2 is highly developmentally regulated among the four isoforms in mESCs, and miR-182 and miR-183 repress AGO2 and regulate stemness in mESCs (Liu and Novak, 2021). Moreover, the AGO2-let-7 interaction downregulates AGO1, a homolog that supports pluripotency, thereby promoting differentiation (Schuh et al., 2025). Despite these critical roles in pluripotency and differentiation, our understanding of how AGO2 is regulated in ESCs is still limited.

Cold Shock Domains (CSDs) are among the most evolutionarily conserved protein domains and are best known for their role in nucleic acid binding (Heinemann and Roske, 2021). Cold Shock Domain-containing protein E1 (CSDE1) is a cytoplasmic RNA-binding protein (RBP) that consists of nine CSDs and plays a critical role in post-transcriptional gene regulation, including translation, mRNA stability, and splicing (Hollmann et al., 2020; Ciocia et al., 2024). CSDE1 was shown to be a component of the miRISC in mESCs and interacts with AGO2 in pluripotent P19 embryonal carcinoma cells, and the miRNA-specific AGO1 in Drosophila Embryo Extracts (Kakumani et al., 2020). Interestingly, in mESCs, CSDE1 destabilizes Gata6 mRNA and prevents mESC differentiation into primitive endoderm (Elatmani et al., 2011). In human ESCs (hESCs), CSDE1 levels are elevated, and CSDE1 knockdown decreased expression of key pluripotency markers, such as OCT4, NANOG, and DPPA2, accompanied by increased neural morphology and increased expression of the neurogenesis transcription factor PAX6 (Ju Lee et al., 2017). Additionally, CSDE1 negatively regulates the mRNA levels and translation of radial glial cell markers FABP7 and VIM, thereby preventing neural fate in hESCs (Ju Lee et al., 2017). Together, these findings highlight CSDE1’s regulatory role in stem cell maintenance. And yet, the molecular understanding of its mechanisms controlling gene expression in embryonic stem cells is incomplete.

In the current study, we examined the role of CSDE1 in regulating AGO2 expression in naïve mouse embryonic stem cells. We demonstrated that CSDE1 influences AGO2 stability and pluripotent protein levels by preventing ubiquitination, and that the N-terminal domain of CSDE1, which is required for AGO2 interaction, is crucial for maintaining AGO2 and pluripotent protein levels. Overall, our findings reveal a new layer of post-translational control over AGO2, mediated by CSDE1 in the regulation of stem cell pluripotency.

Within the miRISC, AGO2 serves as the key RBP that binds miRNAs to target mRNAs for silencing gene expression (Gebert and MacRae, 2019), and CSDE1 interacts with AGO2-miRISC in mESCs (Kakumani et al., 2020). In the current study, we show that CSDE1 stabilizes AGO2 protein (Figures 1A,B) and, consistent with previous studies (Ju Lee et al., 2017), it also influences the expression of pluripotency markers, namely NANOG, SOX2 and Oct4 in mESCs (Figure 1C). However, this relationship is absent in tumour-derived pluripotent P19 EC cells (Supplementary Figure S1), thereby identifying CSDE1 as an upstream regulator of AGO2 and pluripotent proteins in naïve mESCs but not in embryonal carcinomas with stem cell features, likely due to their origin from tumors with malignant properties.

Given that both CSDE1 and AGO2 have been previously implicated in controlling pluripotency in ESCs (Ju Lee et al., 2017; Liu et al., 2024), it is not surprising that CHX treatment led to a marked reduction of CSDE1 within 3 hours, which correlated with the expression of AGO2 and the pluripotent proteins (Figure 2A), suggesting a coordinated regulation of their stability linked to stem cell homeostasis. We also observed a rapid reduction in AGO2 and pluripotent protein levels in CSDE1-depleted mESCs under CHX treatment (Figure 2B), demonstrating that CSDE1 stabilizes AGO2 and pluripotent proteins. Interestingly, in animals, AGO protein degradation occurs through the proteasome and lysosomal pathways (Bressendorff et al., 2025). Accordingly, inhibition of these pathways under CSDE1-depleted conditions restored AGO2 levels in mESCs (Figures 2C,D). Further, findings from the study by Bressendorff et al. identified that the conserved N-coil of AGO2 acts as a structural switch that enables rapid proteolysis (Bressendorff et al., 2025) and, as such, can provide a possible structural basis for CSDE1-mediated regulation of AGO2. Similarly, inhibition of the proteasome (Figure 2E) or the lysosomal (Figure 2F) pathways rescued NANOG, SOX2, and Oct4 expression, suggesting that these factors are degraded via similar mechanisms under CSDE1-depleted conditions, likely involving ubiquitination. While full half-life curve quantification would further strengthen this point, the present data demonstrate directionally consistent effects supporting decreased AGO2 stability via ubiquitination following CSDE1 loss. It presents a coherent model in which CSDE1 loss promotes AGO2- and pluripotent protein-mediated ubiquitination and turnover, and lays the foundation for future detailed biochemical characterization.

Ubiquitin-mediated protein turnover is a fundamental mechanism that maintains stem cell identity by precisely controlling the levels of core pluripotency regulators (Strikoudis et al., 2014). Our observation that CSDE1 and AGO2 are also ubiquitinated in mESCs (Figure 3A) and that loss of CSDE1 enhances ubiquitination of AGO2 and pluripotency factors (Figure 3B) suggests that CSDE1 is an integral component of this post-translational quality-control system. Rather than functioning solely as an RBP, CSDE1 may act as a molecular safeguard that coordinates protein stability and RNA regulatory activity through its interaction with AGO2. This relationship points to a broader model in which the CSDE1–AGO2 axis links ubiquitin-dependent protein quality control with the maintenance of stem cell pluripotency, ensuring that gene expression programs remain tightly regulated during early developmental states.

Consistent with AGO2’s role in sustaining the stem cell transcriptome, we observed that AGO2 knockdown led to reduced expression of NANOG, SOX2, and OCT4 (Figure 1D). Interestingly, this result contrasts with previous studies reporting weaker or even inverse effects of AGO2 loss on pluripotency marker expression (Müller et al., 2020; Liu and Novak, 2021; Liu et al., 2024). A likely explanation for this discrepancy lies in differences in the stem cell lines and culture conditions used. Our experiments employed V6.5 mESCs maintained under serum-free 2iLIF conditions, which support a more naïve pluripotent state with reduced signalling heterogeneity (Silva et al., 2008). In contrast, earlier studies typically used E14 cells grown in serum-containing medium, representing a more primed and heterogeneous state (Liu and Novak, 2021; Liu et al., 2024). These distinct culture environments impose different signalling and metabolic landscapes that can reshape post-transcriptional regulation and protein turnover, thereby influencing how AGO2 controls stem cell gene expression. Thus, the regulatory function of AGO2 in maintaining pluripotency is likely context-dependent, modulated by the pluripotent state and its associated molecular circuitry. Notably, although AGO2 knockdown destabilizes pluripotency factors, CSDE1 levels remain unchanged (Figure 1D), raising the question of whether CSDE1 regulates these proteins directly or via AGO2. Our data point to a requirement for CSDE1–AGO2 interaction in maintaining pluripotency protein stability, as disruption of this interaction through deletion of the CSD1 domain similarly destabilizes NANOG, SOX2, and OCT4 (Figures 4B,C).

In line with previous studies on CSDE1’s role in promoting stemness, overexpression of wild-type CSDE1 increased the expression of NANOG, SOX2 and Oct4, with no significant change in their mRNA levels (Figure 4A; Supplementary Figure S3) (Ju Lee et al., 2017). Conversely, the deletion mutant ΔCSD1 behaved similarly to the vector control. Interestingly, our rescue experiments with AGO2 in CSDE1 knockdown mESCs did not show a robust or consistent restoration of NANOG, SOX2, or Oct4 protein levels (Supplementary Figure S4), indicating that simply increasing AGO2 expression is insufficient to counteract the effects of CSDE1 loss and is consistent with a requirement for functional interaction between CSDE1 and AGO2. This supports the idea that CSDE1 facilitates the recruitment of AGO2 to mRNAs encoding components of the ubiquitin–proteasome system, such as HECW2 (Hynes et al., 2026), thereby indirectly influencing proteasomal degradation of pluripotency regulators.

Given AGO2’s central role in miRNA-guided gene silencing (Gebert and MacRae, 2019), the ubiquitination of AGO2 and its regulation by CSDE1 (Figures 3, 4) likely represent an intersection between post-transcriptional and post-translational control in pluripotent stem cells. By stabilizing AGO2, CSDE1 is well positioned to influence the efficacy and durability of miRNA-mediated gene silencing programs that are essential for maintaining pluripotency. Disruption of this axis may therefore compromise miRNA-dependent repression of differentiation-associated transcripts, linking AGO2 stability directly to stem cell identity. CSDE1 has been implicated in the regulation of mRNA stability and translation of ubiquitin ligases, such as HECW2 (Hynes et al., 2026), suggesting that it may influence protein homeostasis by modulating the ubiquitination machinery. Since CSDE1 interaction with AGO2 via CSD1 is key to maintaining AGO2 stability (Figure 4) (Kakumani et al., 2020), future work using AGO2 miRNA-binding mutants in mESCs will be particularly informative in determining whether the observed post-translational effects depend on miRNA binding of AGO2 or not, as reported by Liu Q et al., 2024, in the case of AGO1 through interactions with heat shock protein Hsp70 and Hsp90 through protein folding. If the loss of the CSDE1–AGO2 interaction derepresses or helps stabilize a ubiquitin ligase targeting AGO2, OCT4, SOX2, or NANOG, it would uncover a feedback mechanism in which CSDE1–AGO2 cooperatively stabilize the stem cell proteome by coupling miRNA function to protein quality control. This model highlights an underexplored layer of gene regulation in which RBPs such as CSDE1 integrate miRNA activity with ubiquitin-dependent proteostasis to sustain pluripotency.