Membraneless cellular condensates, such as P-bodies, represent an important mechanism of spatial organization within the cell, enabling precise regulation of gene expression at the post-transcriptional level. Despite increasing interest in their role, the functional connection between P-bodies and key regulators of cell fate, such as LIN28A, remains insufficiently explored. In this master’s thesis, we investigated the role of cytoplasmic membraneless condensates – P-bodies – in LIN28A-mediated regulation of mRNA stability in mouse embryonic stem cells. To identify interaction partners of LIN28A, we developed and optimized a co-immunoprecipitation protocol that enabled efficient isolation of specific interactions. We found that a buffer containing 0.1–1% Tween 20 and physiological salt concentration allows stable capture of native complexes with minimal nonspecific background, with one hour of incubation being sufficient for adequate yield. Furthermore, we optimized a stepwise gentle elution using sodium citrate, which preserves protein complexes in their native form and is suitable for downstream proteomic analysis. By including benzonase, we demonstrated that most interactions are not RNA-dependent; in some cases, RNA degradation even enabled the binding of additional proteins, suggesting a potential inhibitory effect of RNA on the accessibility of binding sites. Using CRISPR/Cas9 technology, we deleted the DDX6 gene to prevent the formation of P-bodies and introduced inducible expression of LIN28A-GFP. Quantitative immunofluorescence analysis confirmed that LIN28A accumulates in P-bodies only when they are functionally present, indicating that their structure is essential for relocalization upon differentiation signals. Finally, through immunodetection, we showed that P-bodies are required for the stabilization of SOX2, directly confirming that P-bodies are not merely sites of RNA degradation, but spatially organized regulatory hubs necessary for the protection and stabilization of mRNA through LIN28A. Our results provide the first experimental confirmation of a direct role for P-bodies in the stabilization of specific RNA molecules via LIN28A, representing a significant demonstration of their functionality in regulating cell fate. Our findings advance the understanding of spatial post-transcriptional regulation and open avenues for therapeutic development in regeneration, differentiation, and mRNA stability–related diseases, while future studies may further clarify P-body functions in diverse biological contexts.
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