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ANALYSIS OF NON-CANONICAL FUNCTIONS OF THE SELENOCYSTEINE-SPECIFIC ELONGATION FACTOR

by
Aditi Dubey
BS, Drexel University, 2009

Thesis Advisor: Paul Copeland, PHD

Thursday, October 15, 2015
1:00 PM


Abstract

The selenocysteine-specific elongation factor (eEFSec) is a highly specialized factor required for the
insertion of selenocysteine (Sec), the 21st amino acid, into proteins. eEFSec specifically binds and
delivers selenocystnylated-tRNA (Sec-tRNASec) to the ribosome to recode in-frame the UGA
termination codon to Sec codon. This recoding event also requires the presence of a cis stem-loop
structure called a Sec insertion sequence (SECIS) element in the 3 untranslated region (3 UTR) of
the mRNA, bound by the SECIS binding protein 2 (SBP2). The concerted action of these four factors
is required for Sec incorporation in eukaryotes.
eEFSec has several features that distinguish it from the canonical elongation factor eEF1A.
First, eEFSec has a unique Domain IV at the C-terminus that is responsible for the Sec-specific
functions of eEFSec. Domain IV is required for Sec incorporation and is involved in SECIS/SBP2
binding, Sec-tRNASec binding and regulation of GTP hydrolysis. To determine the functions of other
eEFSec domains, a structure-function analysis was performed by swapping domains of eEFSec and
eEF1A and testing the resulting chimeric proteins for Sec incorporation. The chimeric proteins were
inactive for Sec incorporation, indicating that the appropriate structural context is insufficient for
activity. Another feature that contrasts eEFSec from eEF1A is that it localizes to the nucleus while
eEF1A does not. Analysis of sequences unique to eEFSec identified several residues that are critical
for nucleocytoplasmic shuttling of eEFSec. However, blocking nuclear export of eEFSec did not alter
endogenous selenoprotein expression, indicating that nuclear localization of eEFSec is not required
for selenoprotein production. Finally, unlike eEF1A, eEFSec is not known to interact with a gunanine
exchange factor (GEF) during GTP hydrolysis in bacteria or archea. To determine if eEFSec requires
a GEF in mammals, immunoprecipitation experiments were conducted. eEFSec was not found to
interact with any GEF or GEF-like proteins. Instead, it interacts with nucleolar proteins nucleophosmin
(NPM1) and DEAD-box polypeptide 21 (DDX21) that are involved in ribosomal biogenesis. Further
studies are needed to determine the role of nuclear eEFSec and the significance of these
interactions.


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