Determination of molecular mechanism of SAMD9 for recognition and cleavage of phenylalanine tRNA by tRNase SA

SAMD9 is a human protein associated with innate immunity and disease suppression, including roles in inflammatory disorders and antiviral responses. Recent findings suggest that a truncated fragment of SAMD9 (residues 156–385) possesses enzymatic activity toward phenylalanine tRNA (tRNAPhe), classifying it as a tRNase with potential anticodon nuclease (ACNase) function. To investigate this, a recombinant His-SUMO-tagged SAMD9 (156–385) construct was expressed in E. coli and purified using Ni-NTA affinity chromatography, ULP-1 protease cleavage, and size exclusion chromatography. SDS-PAGE analysis confirmed successful expression and isolation of the protein in a soluble, monomeric form. A final concentration of 25.16 mg/mL was achieved, indicating high yield and purity. Binding of SAMD9 to tRNAPhe was assessed using a native gel shift assay, comparing wild-type and W333A mutant proteins. Both variants formed protein-RNA complexes, as evidenced by mobility shifts in native PAGE, suggesting preserved tRNA-binding activity. These findings provide foundational insight into the RNA recognition and cleavage capabilities of SAMD9 and lay the groundwork for future studies into its enzymatic mechanism and role in innate immune regulation.