Cytoplasmic poly(A)-binding proteins (PABPCs) are essential and highly abundant regulators of mRNA stability and translation, but their cellular functions have been difficult to dissect due to slow turnover and the lethality observed upon loss of both major paralogs, PABPC1 and PABPC4. To enable structure-function analysis of PABPC in human cells, we developed a protein-replacement platform that couples rapid auxin-inducible degradation of endogenous PABPC1 and PABPC4 with doxycycline-controlled expression of engineered PABPC variants. This approach enables acute removal of native PABPC and real-time assessment of how specific domains, paralogs, and sequence alterations support cellular fitness, shape transcriptome profiles, and regulate poly(A)-tail length. Using this system, we show that the RRM4 domain of PABPC1 is essential for growth, whereas post-translationally modified lysines within RRM4 are individually dispensable. Paralogs and variants with heterologous RRM4 domains vary in their ability to substitute for PABPC1, revealing functional divergence among PABPCs. Transcriptome profiling identifies variant-specific regulatory signatures, and dose-controlled rescue further delineates the relationship between PABPC variant abundance and global poly(A)-tail lengths in vivo. Together, this platform provides a generalizable strategy for dissecting PABPC biology in the cellular context using rationally designed variants.
Muller, R. Y., Wang, K. X., Bartel, D. P.
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