Towards an integrated in vivo model of translational dysfunction and tissue specific phenotypes in ribosome disease
Keywords: Genetics.

In the 1930s, Josephs, Diamond, and Blackfan described patients with an unusual, highly selective congenital defect in hematopoiesis that presented with pure red blood cell aplasia without affecting other hematopoietic lineages (Diamond et al., 1961). This syndrome was later shown to be associated with a number of other developmental defects, including impaired limb patterning, craniofacial morphogenesis, and heart and urogenital development (Da Costa et al., 2018). It wasn't until 1999 that one cause of "Diamond-Blackfan Anemia" (DBA) was shown surprisingly to be deficiency of a small subunit ribosomal protein RPS19, an essential component of the ribosome (Draptchinskaia et al., 1999). Since then, haploinsufficiency of ~20 different ribosomal proteins (RPs) have been implicated in DBA, suggesting that core dysfunction of a machine thought to be as ubiquitous and essential as the ribosome can somehow mysteriously lead to a common set of tissue specific phenotypes (Da Costa et al., 2018). However, despite work in the past 20 years, it is still unclear how these in vivo phenotypes emerge in these "ribosomopathies." Here, I describe work towards understanding how ribosome dysfunction leads to specific developmental phenotypes. Chapter 1 provides a brief introduction to the ribosome and to human diseases of the ribosome encompassed by the ribosomopathies. This chapter provides a description of the current understanding of the field and highlights the many unknowns for how ribosome dysfunction can lead to tissue specific disease. To date, the phenotypes underlying ribosomopathies are primarily thought to arise from translation-independent processes driven by activation of the stress-induced transcription factor p53, which in turn transcriptionally activates target genes canonically involved in regulating cell proliferation and apoptosis (Farley-Barnes et al., 2019; Fumagalli and Thomas, 2011; Narla and Ebert, 2010). However, how such generic effects on cells lead to highly specific developmental phenotypes has been a paradox. Furthermore, although one would expect translational regulation to be perturbed in ribosomopathies, it is thus surprising that changes to translation have been largely unexplored, especially within relevant in vivo developmental contexts given the tissue specific phenotypes observed. As a result, in Chapter 2, I describe work to systematically dissect translational changes upon haploinsufficiency of an essential RP in vivo. Because ribosomopathies can manifest with specific limb developmental phenotypes, we use the developing limb as a model to probe developmental phenotypes that result from RP haploinsufficiency. Combining comprehensive mouse genetics and in vivo ribosome profiling, we observe limb patterning phenotypes in RP haploinsufficient embryos and uncover selective translational changes of transcripts controlling limb development. Surprisingly, we discover that many of the translational changes that emerge from RP haploinsufficiency do not emerge directly from ribosome dysfunction but rather emerge through a regulated pathway mediated by p53 itself. This led to identification of an unappreciated role of p53 as the mediator between ribosome dysfunction and translational change as well as a master regulator of protein synthesis through transcriptional activation of 4E-BP1, a key repressor of cap-dependent translation (Lin et al., 1994). Given that we observed limb developmental defects and translational perturbation of transcripts involved in limb patterning, we next asked how RP haploinsufficiency impinges on well-established developmental patterning pathways in Chapter 3. We discover that the Sonic hedgehog pathway (Shh), the master pathway responsible for patterning the limb along the anteroposterior (AP) axis, is perturbed in the limb. These results are consistent with selective AP patterning defects in RP haploinsufficiency embryos, such as loss of the anterior radial bone in the limb. Finally, in Chapter 4, I describe future directions and unanswered questions in understanding the mechanisms underlying ribosome disease. Together, these chapters elucidate how translational dysregulation emerges from general ribosome dysfunction in vivo and lays the foundation for understanding how such dysregulation leads to tissue specific patterning phenotypes in ribosomopathies