Poster abstracts

Poster number 53 submitted by Roshan Mascarenhas

Precursor-product relationship in the erythroid cell-specific decay of nonsense-containing β-globin mRNA

Roshan Mascarenhas (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210), Julie A. Dougherty (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210), Daniel R. Schoenberg (Center for RNA Biology, Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
In erythroid cells endonuclease cleavage is the rate-limiting step in the decay of nonsense-containing human β-globin mRNA. This appears to be a stochastic process as there is no evidence for any order of cleavage site detection and origin of stable decay products. The 5’-truncated intermediates generated during the decay process are polyadenylated and more stable than the parent mRNA. This complicated the approaches for studying the precursor-product relationship of these to full-length mRNA. In addition we needed a more sensitive and facile assay to monitor the decay process than the S1 nuclease protection assay that was used in our previous work. To address this we developed lines of K562 cells that are stably transfected with tetracycline-inducible forms of wild-type and PTC-containing human β-globin genes. These were used to quantify the precursor-product relationship of full-length mRNA and an endonuclease decay product using the newly-developed MBRACE assay. MBRACE is based on ligation of a primer onto the RNA 5’ end and amplification of the resulting product using primers to the different 5’ junctions and a 3’ globin specific primer. Using this we confirm the precursor-product relationship of full-length PTC-containing β-globin mRNA to the 5’-truncated decay product, and examined the role of a stabilizing element in the 3’-UTR in the prolonged half-life of the decay product.

Supported by PHS grant GM079707

Keywords: Nonsense-mediated decay, beta-globin mRNA