An entire class of human diseases, collectively referred to as """"""""ribosomopathies"""""""", is associated with mutations in distinct components of the ribosome machinery. An outstanding question is how """"""""defected"""""""" ribosomes produce specific pathological features. An important example is X-linked Dyskeratosis Congenita (X-DC), which is invariably associated with mutations in the DKC1 gene encoding for an enzyme that modifies ribosomal RNA (rRNA). X-DC is associated with specific pathological features, including bone marrow failure and increased cancer susceptibility. We engineered DKC1 hypomorphic mice (DKC1m) that faithfully recapitulate the disease. In the previous funding period, we developed an unbiased polysome microarray translational screen and uncovered the first functional targets of X-DC that include important mRNAs such as p53 and p27. Strikingly, these target mRNAs share a common regulatory motif in their 5'untranslated region known as an internal ribosome entry site (IRES) element. Our preliminary data strongly suggest that defects in this mode of translation can underlie, at least in part, specific pathological features of X-DC. The studies tha we seek to address in the current proposal will significantly extend our understanding of X-DC and ribosomopathies by providing unique mechanistic insight into how deregulations in translational control lead to a specific disease state.
In Aim 1, we will undertake a genetic approach to assess in vivo how impairments in rRNA modifications lead to specific alterations in oncogene induced senescence, a critical and early barrier in cancer development. We will also employ a unique RNA affinity purification protocol to molecularly and biochemically characterize the mechanisms by which impaired rRNA modifications affect p53 IRES- dependent translation.
In Aim 2, we will characterize the role of DKC1 and translational control in the maintenance of bone marrow stem cell quiescence, a process that may be critically deregulated in X-DC leading to bone marrow failure, one of the major causes of patient mortality. Lastly, in Aim 3 we will employ a unique collection of human X-DC patient cells as well as X-DC induced pluripotent cells to delineate the contribution of specific point mutations in the DKC1 gene towards translational control and hematopoietic development, thereby defining the genotype to phenotype landscape of X-DC pathogenesis. Importantly, we have developed a novel technology known as ribosome profiling that will enable us to delineate the translational landscape of the X-DC patient genome at a codon-by codon resolution. The proposed studies will be instrumental to our understanding of how ribosome impairments give rise to specific pathological features of X-DC. Moreover, they will also extend our general understanding of the molecular basis for ribosomopathies. )

Public Health Relevance

An entire class of fatal inherited human syndromes such as X-linked Dyskeratosis Congenita (X-DC), cartilage- hair hypoplasia (CHH), Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome (SD), and 5q- syndrome are associated with mutations to the cellular machinery responsible for protein production. The goal of this proposal is to study X-DC as a paradigm for these diseases;specifically, to understand the mechanisms by which deregulation in protein production lead to bone marrow failure and cancer susceptibility, the most common and severe symptoms suffered by afflicted patients. Our important findings will provide the rationale for a new line of therapeutics not only for X-DC, but also for a broad scope of life threatening diseases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Therapeutic Approaches to Genetic Diseases (TAG)
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Bishop, Terry Rogers
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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