Fig. 1
Identification of a putative tsRNA-binding protein in Fn. a Illustration of the workflow for an RNA affinity pulldown assay to identify RNA-binding proteins. 5′ or 3′ biotinylated RNA oligonucleotides are immobilized on the surface of streptavidin-conjugated paramagnetic microparticles to capture RNA-interacting proteins from cell lysate. b Silver staining of proteins from the biotinylated RNA pulldown in Fn ATCC 23726. Results are representative images of four independent experiments. c Heatmap showing the relative abundance of proteins isolated from the gel band at 75 kDa through mass spectrometry. The most significant band labeled as ‘D5RD38’ (P-type ATPase transporter, PtaT) was highlighted by a red arrow. d Validating the interaction between biotinylated tsRNA and recombinant PtaT. 5′ or 3′ biotin tsRNA-mediated affinity pulldown was performed using the total lysate of E. coli BL21 Rosetta, in which FLAG-tagged PtaT was recombinantly expressed. 5′ and 3′ biotinylated scrambled RNA serves as a negative control. The lower panel (input) represents an equal amount of total lysates used, and the upper panel (pulldown) indicates the amount of FLAG-tagged PtaT specifically interacting with different biotinylated RNA. A representative image of two independent experiments is shown. e Purified recombinant FLAG-PtaT can specifically bind tsRNA-000794 and tsRNA-020498 but not their DNA counterparts. Recombinant FLAG-tagged PtaT, which was purified from E. coli BL21 Rosetta, was directly used for the pulldown assay with 5′ biotinylated tsRNA. 5′ biotinylated scrambled RNA and beads only served as the negative control
