Dissecting the tRNA Fragment tRF3E-Nucleolin Interaction: Implications in Breast Cancer

Nucleolin (NCL), an RNA-binding protein which regulates critical cellular processes, is frequently dysregulated in human cancers, including breast cancer, making it an attrac-tive therapeutic target. However, molecular details of the RNA-NCL interaction have not been investigated yet. A tRNA fragment named tRF3E, displaying tumor suppressor roles in breast cancer, was found to bind NCL with high affinity displacing NCL-controlled transcripts. Here, we investigated the determinants and cooperativity of tRF3E-NCL in-teraction by Electrophoretic Mobility Shift Assays and in silico docking analysis, using wild-type or mutated tRF3E. We found that NCL, through its RNA-binding domains (RBD1-2 and RBD3-4), binds simultaneously two tRF3E molecules, giving rise to an en-ergetically favored complex. Instead, a mutant form of tRF3E (M19-24), in which the NCL recognition element in position 19-24 has been disrupted, contacts NCL exclusively at RBD3-4, causing the loss of cooperativity among RBDs. Importantly, when expressed in MCF7 breast cancer cells, tRF3E significantly reduced cell proliferation and colony for-mation, confirming its role as tumor suppressor, but tRF3E functional properties were lost when the 19-24 motif was mutated, suggesting that cooperativity among multiple domains is required for the NCL-mediated tRF3E antitumor function. This study sheds light on the dynamic of RNA-NCL interaction and lays the foundations for using tRF3E as promising NCL-targeted biodrug candidate.