RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors
(A) Phylogenetic distribution of RsfA (Interpro entry IPR004394 [DUF143]) and ribosomal protein L14 (IPR000218) on the iTOL tree of life . Triangles indicate species in which the RsfA-L14 interaction was detected by binary detection assays (grey), co-purification with the LRS (white) or both (black). Known RsfA-L14/LRS interactions are listed in Table S1. (B) T. pallidum RsfA (TP0738) interacts strongly with L14 (TP0199) and very weakly with other proteins involved in translation  in yeast-two-hybrid assays. C, control (with empty prey vector to measure self-activation of the bait). This interaction is also conserved in E. coli (C). (D, E) RsfA and L14 homologues from human and maize interact in pull down experiments. RsfA homologues were tagged with NusA-His6 (N) and L14 homologues with maltose binding protein (M) (human mtRsfA = C7orf30, mitochondrial ribosomal protein L14 = L14mt; maize RsfA = Iojap, maize chloroplastic L14 = RPL14); i = input samples, o = output samples. Constructs with the corresponding Interpro signatures and the range of cloned codons are illustrated on the right. (F) Human mitochondrial C7orf30 (mtRsfA) co-localizes with L14mt exclusively into mitochondria as visualized by MitoTracker Green. Nuclei visualized by DRAQ5 (blue) and membranes by eCFP-membrane (cyan). Co-localization of both mtRsfA (C7orf30) and L14mt in mitochondria is indicated in yellow. (G) Bi-molecular fluorescence complementation (BiFC) reveals the interaction of mtRsfA (C7orf30) and L14mt in mitochondria. Overlay images represent DRAQ5 (blue), CFP-membrane (cyan) and BiFC stained cells. Green fluorescence indicates interaction-dependent regeneration of the Venus protein. Constructs are shown below. Here, the hexagons symbolize the native N-termini including mitochondrial localization sequences.