? The long-term objective of this proposal is to elucidate the molecular and cellular basis for X-linked Dyskeratosis Congenita (X-DC), an inherited disease characterized by early mortality due to bone marrow failure as well as increased tumor susceptibility. The gene found mutated in X-DC, Dyskeratosis Congenita 1 (DKC1), is a pseudouridine synthase that mediates post-transcriptional modification of ribosomal RNA (rRNA) of both large and small ribosomal subunits through conversion of the uridine nucleotide (U) to pseudouridine (psi). The role of rRNA modifications in ribosome function and protein synthesis is poorly understood. We have generated DKC1 hypomorphic mice (DKC1m), which faithfully recapitulate all the clinical features of X-DC and are the first mouse model for severe aplastic anemia, a life threatening disease present in a heterogeneous group of disorders characterized by the failure in forming blood cells. DKC1m mice, also show increased cancer susceptibility and more than 50% of these animals develop carcinomas and B-cell lymphomas. We have shown that primary cells from DKC1m mice possess impairments in rRNA pseudouridylation and display decreased ribosome activity prior to disease onset. As outlined in the preliminary studies section, we have successfully utilized a proteomics strategy to identify a subset of mRNAs that are translationally impaired in DKC1m cells. These findings support a role for rRNA modifications in translation initiation of specific mRNAs, which harbor an internal ribosome entry site (IRES) positioned in their 5'UTR (5'untraslated region). The goals of the proposed study are to investigate the role of dyskerin and rRNA modification in control of protein synthesis. In addition, utilizing a molecular and genetic approach we will monitor translation regulation in vivo in DKC1m mice and genetically test the functional consequences of translational impairments in key target mRNAs towards the pathological features of X-DC.
In Aim 1 we will expand on our proteomics strategy to characterize mRNAs, which rely on dyskerin activity and rRNA modifications for translation initiation.
In Aim 2 we will define, in vivo, the role of defective IRES-dependent translation in X-DC pathogenesis utilizing a genetic approach as well as a novel live imaging system to follow IRES-dependent translation. Finally, in Aim 3 we will determine the molecular step(s) in which DKC1m ribosomes fail to initiate protein synthesis utilizing an in vitro reconstituted system. Lay abstract. Together these studies will build a deeper foundation for understanding the key genetic and molecular events that cause X-DC human disease and provide new animal models that will be vital for understanding the underlying causes of bone marrow failure and cancer susceptibility. ? ? ? ? ? ?

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GTIE-A (01))
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Mitchell, Phyllis
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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