The beta-thalassemias are common genetic disorders that result from mutations in the beta globin gene. Many of these mutations introduce a premature termination codon (PTC) that results in the degradation of mRNA encoded by the affected allele. Individuals inheriting mutations in both beta-globin alleles have severe anemia, hemolysis, and secondary pathology resulting from expansion of the bone marrow. Most PTC containing mRNAs are degraded through a nucleus-associated surveillance pathway termed nonsense mediated mRNA decay (NMD). Previous work demonstrated that a PTC in exon 2 of the human beta-globin gene activated the cytoplasmic degradation of beta-globin mRNA, with the production of metastable decay intermediates. We replicated this in a model system using murine erythroleukemia cells, and determined that mRNA decay results from endonucleolytic cleavage. The degradation of both normal and PTC-containing beta-globin mRNA is catalyzed by a polysome-associated ribonuclease whose properties are similar to a polysome-associated endonuclease identified in Xenopus termed xPMR1. We propose that the endonuclease-catalyzed degradation of PTC-containing beta-globin mRNA in erythroid cells is a specialized form of NMD.
Aim 1 will use transcription pulse-chase and a new, sensitive FRET-based assay to examine details of the mRNA decay process and how they relate to structural features of beta-globin mRNA.
Aim 2 will examine the relationship between the PTC-stimulated degradation of beta-globin mRNA and NMD by RNAi of Upf1, expression of dominant negative proteins, examining the relationship of this process to downstream intron splicing, and by examining its relationship to the pioneer round of translation.
Aim 3 will characterize the beta-globin mRNA, focusing on a recently identified unique gene believed to encode this enzyme. This will entail expression of recombinant protein, characterization of its biochemical properties, and experiments studying the impact of inactivating mPMR1 by RNA interference on the PTC-stimulated degradation of beta-globin mRNA. The long-term goal is to identify new therapeutic targets for treating beta-thalassemia and other diseases caused by PTCs by better understanding the enzymatic mechanisms responsible for the degradation of PTC-containing mRNAs.
| Otsuka, Yuichi; Kedersha, Nancy L; Schoenberg, Daniel R (2009) Identification of a cytoplasmic complex that adds a cap onto 5'-monophosphate RNA. Mol Cell Biol 29:2155-67 |
