Ribosomal proteins (RP) are increasingly understood to play crucial regulatory roles in development and disease; however, the way they do so remains unclear. The potential modes of RP action fall into two classes: effects on the ribosome itself or extraribosomal functions carried out while physically separate from the ribosome. Traditional loss-of-function approaches to address RP function fail to distinguish these modes of action because most RP are essential for ribosome biogenesis and/or function and so ablation of such RP genes attenuates both modes of action. Consequently, an unequivocal determination of the contributions of extraribosomal RP functions to biological processes remains a critical gap in knowledge. Importantly, we have identified an RP, Rpl22, which is not required for ribosome biogenesis or function, but nevertheless plays critical, selective roles in hematopoiesis. Rpl22 not only regulates hematopoietic stem cell (HSC) emergence in the embryo but also regulates adult HSC function, as well as more distal stages of hematopoiesis including B and T lymphocyte development. The basis for the selective requirement for Rpl22 in particular hematopoietic processes remains unclear; however, we have recently determined that the capacity of Rpl22 to regulate fetal HSC emergence is dependent upon its physical association with hnRNP-A1, which can regulate translation. Moreover, hnRNP-A1 selectively associates with Rpl22 molecules that are physically separate from the ribosome. Consequently, we have an unprecedented opportunity to investigate the importance of the extraribosomal function of Rpl22 in hematopoiesis. We hypothesize that Rpl22 regulates hematopoiesis chiefly through its extraribosomal activity, by associating with hnRNP-A1, and regulating the translation, splicing and possibly the stability of RNA targets that differ depending on the cellular context. Indeed, while Rpl22 controls fetal HSC emergence by regulating the expression of Smad1, Rpl22 controls adult HSC behavior by regulating the expression of a lipoxygenase (Alox12), which generates lipid species that activate PPARd, a master regulator of fatty acid oxidation (FAO). As FAO has been shown to regulate HSC self-renewal, we hypothesize that Rpl22 regulates Alox12 signaling to maintain HSCs. In this proposal, we will test these hypotheses in two aims.
Aim 1 seeks to determine how the Rpl22/hnRNP-A1 axis exerts its functions in controlling HSC biology, and identify the specific Rpl22-regulated fetal and adult hematopoietic processes that depend on Rpl22 association with hnRNP-A1.
Aim2 focuses on investigating the mechanism and role of downstream Alox12-driven FAO pathways through which Rpl22 controls adult HSC function. In pursing these studies, we will integrate cutting edge structural analysis with the unique strengths of zebrafish and a variety of novel mouse models, and verify core findings in human progenitors. Together, these studies will provide the first insight into the novel, extraribosomal ?moonlighting? functions through which RP regulate biological processes, and may reveal the basis for the tissue specificity of Rpl22- dependence displayed by particular hematopoietic processes.
- HEALTH RELEVANCE Gaining insight into the capacity of Rpl22 to regulate HSC function through effects on metabolism will inform future efforts to devise novel strategies to exploit fatty acid oxidation (FAO) as a therapeutic vulnerability in acute myelogenous leukemia and other hematologic malignancies. In addition, these studies stand to open up new avenues for combinatorial use with cancer immunotherapy, as inhibition of FAO has been demonstrated to synergize with checkpoint blockade.
