The C-terminal region of TENT5 proteins drives ER-associated mRNA polyadenylation via FNDC3 interaction.

Spatial regulation of mRNA polyadenylation emerges as a key mechanism shaping cellular function. The TENT5/FAM46 family comprises four non-canonical poly(A) polymerases that stabilize transcripts encoding ER-targeted proteins, with mutations linked to diseases of professional secretory cells. Using transcriptomic and proteomic profiling with systematic mutagenesis, we show how paralog-specific divergence drives distinct localization, interactions, and functions. TENT5D, the most ER-associated member, remodels the proteome, enhancing the expression of ER, ERGIC, Golgi, and lysosomal proteins. In contrast, TENT5B lacks ER targeting and regulates proteins involved in cell division. A member-specific C-terminal region that binds ER-transmembrane FNDC3 proteins is necessary and sufficient for ER localization. Mutations in this region of TENT5C, found in multiple myeloma, impair FNDC3 binding or stability, reducing immunoglobulin production and tumor-suppressive activity. Overall, we define a domain-encoded mechanism linking TENT5 localization to transcript selectivity and secretory output, with direct implications for compartmentalized mRNA regulation and development of RNA-based therapeutic strategies.