 | Nianest Alers University of Puerto Rico, Aguadilla BiologyMentor(s)Paul Copeland, Ph.D. Jesse Donovan Kelvin Cabán Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School |
| ||
| Characterization of a putative negative regulatory element in the Sel P 3’UTR | ||
| Selenium is an essential component of several metabolic pathways and is found in proteins as the amino acid selenocysteine (Sec). Sec incorporation occurs at in-frame UGA codons and this requires a Selenocysteine insertion sequence (SECIS) element located in the 3’ untranslated region (UTR) of the mRNA. Two trans-acting factors are also required, a SECIS binding protein, and a Sec-specific elongation factor. Interestingly, the mRNA encoding the protein selenoprotein P (Sel P) has ten in-frame UGA codons and two SECIS elements in its 3’ UTR. Sel P exists in several isoforms in vivo due to termination at several of its UGA codons. In addition to the SECIS element, the Sel P 3’UTR has a highly conserved sequence (nt 1-74) in its 5’ end. To determine the functional relevance of this region in Sec incorporation, we cloned the Sel P 3’UTR into a luciferase construct containing an in-frame UGA codon and two constructs containing a deletion of nt 1-74 (Δ 1-74) and nt 1-273 (Δ 1-273) in the Sel P 3’UTR. mRNAs corresponding to these constructs were translated in Rabbit Reticulocyte Lysate (RRL) in the presence of 35S-Met and Sec incorporation activity was measured by the ability to produce full-length luciferase. Deletion of nt 1-74 results in a 2-fold increase in total protein translation but not in Sec incorporation efficiency, which indicates that this conserved region has a repressive effect on translation. Surprisingly, deletion of nt 1-273 has wild type levels of total protein translation indicating that there may exist a positive regulatory element between nt 74 -273. We are currently performing RT-PCR to check if this result is due to differences in RNA stability. To determine if the sequence 1-74 has a repressor function on its own, we are also cloning its DNA into the native luciferase 3’ UTR. We speculate that nt 1-74 performs a negative regulatory function by serving as a docking site for other trans factors that modulate the amount of Sel P isoforms synthesized. |