OCR Text |
Show THE UNIVERSITY OF UTAH UNDERGRADUATE RESEARCH ABSTRACTS Functional Characterization of a Highly Conserved RNA Structure Bryndon B. Hatch, Christine B. Anderson (Michael T. Howard) Department of Human Genetics University of Utah Proteins containing selenium in the form of the 21st amino acid, selenocysteine, perform important enzymatic oxidation-reduction reactions in the body. Expression of these selenoproteins requires that a UGA codon in the mRNA, normally a stop codon, be redefined to encode the amino acid selenocysteine. Proper expression and functionality of these selenoproteins is required for normal muscle development and maintenance, thyroid hormone metabolism, fertility, and immune function. Mutations in the genes that encode for these proteins have been linked with certain inherited human myopathies, such as Rigid Spine Muscular Dystrophy and Multiminicore disease. The research being performed is designed to determine if a highly conserved stem loop structure located near the UGA selenocysteine codon within the Sps2 mRNA has an important function in the redefinition process. Mutations in the Sps2 gene were designed to test the functionality of various parts of the predicted stem loop structure. The mutations were produced by performing multi-stage PCR and were inserted into a dual luciferase reporter plasmid, p2luc. The vectors were then be cloned and then transfected into cultured mammalian cells to perform translation of the reporters. The p2luc dual luciferase reporter plasmid was used to quantify the expression levels of SPS2 proteins. The vectors contain two reporters, one of which requires redefinition of the UGA codon for expression. The ratio of the downstream reporter activity compared to the upstream reporter activity is proportional to the degree of selenocysteine insertion. Mutations in the Sps2 stem loop structure altered the levels of selenocysteine insertion in an unexpected way. Two of the clones, which caused mismatches in the four nucleotides at the base of the stem loop, increased levels of selenocysteine insertion. This increase is indicative of increased functionality of the stem loop structure. Further tests will be designed to test whether the increased distance of the stem loop from the UGA stop codon, decreased length of the stem loop, or closer proximity to another stem loop in the mRNA is responsible for the increased functionality of the stem loop. Bryndon B. Hatch Michael T. Howard 32 |