The P1/ P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells.

Ricin A-chain ( RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α- P2β and P2α- P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the sarcin-ricin loop. Using small interfering RNA-mediated knockdown of P1/ P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/ P2-depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1- P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes. Structured digital abstract P2, P1 and RTA physically interact by surface plasmon resonance (View interaction), P2, P1 and RTA physically interact by anti bait coimmunoprecipitation (View interaction) [ABSTRACT FROM AUTHOR]