Poster abstracts

Poster number 25 submitted by Eric Cockman

Elements in the 3’UTR of Selenoprotein S regulate Selenocysteine insertion

Eric Cockman (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH), Jodi Bubenik (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH), Donna Driscoll (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH)

Abstract:
Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved at the translational level by the recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNA transcripts possess a 3’UTR sequence, known as the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. The SECIS elements of selenoproteins differ in sequence and structure and vary in efficiency of Sec insertion (1). The dogma in the field is that the SECIS element is the major RNA sequence that determines Sec incorporation. However, our lab has shown that the full 3’UTR of Selenoprotein S (SelS) has a much lower recoding efficiency than the SelS SECIS alone, suggesting that there are other elements in the 3’UTR that regulate the insertion of Sec (2).
Using a luciferase reporter assay in rabbit reticulocyte lysate (RRL), we have shown that a deletion of the last 101 nucleotides of the SelS 3’UTR restored Sec incorporation efficiencies to the level of the SelS SECIS alone. Alignments of this region across species revealed three conserved sequences. Deletion of the distal most sequence rescued recoding efficiency of the SelS 3’UTR in the RRL system. Using UV-crosslinking, we observed the binding of two proteins, approximately 40-45 kDa in size, that are present in multiple cell types. Based on competition experiments, the two proteins require the distal most sequence for high affinity binding. Thus, the protein binding correlates with the recoding results. Currently, RNAase-assisted affinity purification is being performed to isolate and identify the binding proteins.

References:
1) Latreche L, Jean-Jean O, Driscoll DM, Chavatte L (2009) Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine. Nucleic Acids Res 37: 5868–5880gkp635
2) Bubenik JL,Miniard AC, Driscoll DM , Alternative Transcripts and 3’ UTR Elements Govern the Incorporation of Selenocysteine into SelenoproteinS PLoS One. 2013 Apr 16;8(4):e62102. doi: 10.1371/journal.pone.0062102. Print 2013

Keywords: Selenoproteins, RNA-Protein Interaction, Selenocysteine