Regulation of hematopoiesis by ribosomal protein paralogs

PROJECT SUMMARY (See instructions): Ribosomal proteins (RP) play crucial regulatory roles in development and disease, but the way they do so remains unclear and is hotly debated. The regulatory functions of RP have been suggested to be mediated through two distinct modes of action: effects at the ribosome itself, or extraribosomal functions performed while physically separate from the ribosome. It is exceedingly difficult to definitively distinguish these modes of action because traditional loss-of-function approaches abrogate both modes of action. Consequently, an unequivocal determination of the contribution of extraribosomal RP functions to biological processes remains to be achieved, and this represents a critical gap in knowledge. Filling this key gap was the subject of our original proposal, which provided the first in vivo evidence for extraribosomal activity of an RP in controlling development and disease. Specifically, we showed that the RP, Rpl22 is not required for ribosome biogenesis or function, but nevertheless plays critical and selective roles in hematopoiesis while physically separate from the ribosome, in association with the cofactor hnRNP-A1. Rpl22 is thus an outstanding exemplar of an RP with extraribosomal functions, and examining its biology will illuminate how RP engage in signaling pathways beyond the ribosome. We have generated mutant mice (Rp/22dN mutant) that break the Rpl22-hnRNP-A1 interaction and these mice have revealed even more interesting findings. Specifically, Rp/22LJN mutant mice do not display the baseline defects in hematopoiesis observed in mice lacking the entire Rpl22 protein. Instead, these mice exhibit an inability to restrain dysplastic hematopoietic processes. Thus, these findings provide the first evidence for a duality of extraribosomal function, where Rpl22 can act to both promote and restrain hematopoiesis in different contexts. Consequently, we hypothesize that extraribosomal Rpl22 exists as genetically separable complexes, one that contains hnRNP-A1 and restrains hematopoiesis, and another of completely unknown composition that promotes hematopoiesis. As Rpl22 is an RNA binding protein, we additionally hypothesize that each of these complexes mediate their effects by associating with distinct pools of cellular mRNA. Our goal is to elucidate the nature of these distinct extraribosomal complexes (Aim1) and identify the mRNA targets through which they mediate their support and restraint of hematopoiesis (Aim2). These studies promise to provide paradigm altering insight into the nature and mechanistic basis for extraribosomal functions in development and disease.