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Post-transcriptional regulation of selenoprotein expression by SECIS binding proteins

by
Jesse Donovan
B.S., The College of New Jersey 2005

Thesis Advisor: Paul R. Copeland, Ph.D.
Graduate Program in Molecular Genetics, Microbiology & Immunology

RWJMS,Room V-14
Piscataway

Wednesday, May 18, 2011
1:00 p.m.


Abstract

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at specific UGA codons. Recoding UGA from stop to Sec requires a cis-acting stem-loop structure, termed the SECIS element, in the 3 untranslated region of selenoprotein mRNAs. SECIS binding protein 2 (SBP2), Sec-tRNASec, and the Sec specific translation elongation factor, eEFSec, are also essential. Additionally, there is a homologue of SBP2, termed SBP2L, that has no known function. The work presented herein describes the characterization of a domain in SBP2 and preliminary evidence that SBP2L regulates selenoprotein expression.
Mammalian SBP2 is composed of an N-terminal domain of unknown function, a central Sec incorporation domain (SID), and a C-terminal RNA binding domain (RBD). Multiple sequence alignments were used to design mutants in the SID. Analysis of these mutants in Sec incorporation assays, SECIS binding and ribosome binding assays, revealed residues critical for Sec incorporation without defects in SECIS or ribosome binding, suggesting they function downstream of SECIS binding. Expressing the SID and RBD as separate recombinant proteins and showed that when combined they can promote Sec incorporation and SECIS binding to levels comparable to intact SBP2. Co-immunoprecipitation studies showed a SECIS element dependent interaction between the SID and RBD, which suggests that a conformational change occurs in SBP2 upon binding SECIS RNA.
Bioinformatic analysis of SECIS binding proteins showed that SBP2L is a paralogue of SBP2 in vertebrates and that the genomes of invertebrate deuterostomes encode SBP2L as their only SECIS binding protein. This suggests SBP2L from these species can promote Sec incorporation while human SBP2L is unable to do so. This led to the hypothesis that SBP2L may preferentially bind a subset of SECIS elements. The apparent Kd of all human SECIS elements for SBP2 and SBP2L was determined and it was found that SBP2L had the highest affinity for the selenoprotein V SECIS element, yet this SECIS element was not capable of promoting SBP2L-dependent Sec incorporation. Nonetheless, selenoprotein mRNAs co-immunoprecipitated with SBP2L from PC3 and U87MG cell extracts, implicating SBP2L in the regulation of selenoprotein expression.


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