Nucleic Acids Research, 2009, Vol. 37, No. 17Published online 3 August 2009
Novel structural determinants in human SECIS
elements modulate the translational recoding
of UGA as selenocysteine
Lynda Latre `che1,2, Olivier Jean-Jean2, Donna M. Driscoll3,4and Laurent Chavatte1,3,*
1Centre de recherche de Gif-sur-Yvette, FRC 3115. Centre de Ge ´ne ´tique Mole ´culaire, CNRS, FRE 3144,
Gif-sur-Yvette,2UPMC Univ Paris 06, FRE 3207, CNRS, F-75005 Paris, France,3Department of Cell Biology,
Lerner Research Institute, Cleveland Clinic Foundation and4Department of Molecular Medicine, Cleveland Clinic
Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
Received June 8, 2009; Revised July 15, 2009; Accepted July 16, 2009
The selenocysteine insertion sequence (SECIS)
element directs the translational recoding of UGA
as selenocysteine. In eukaryotes, the SECIS is
located downstream of the UGA codon in the
3’-UTR of the selenoprotein mRNA. Despite poor
sequence conservation, all SECIS elements form a
similar stem-loop structure containing a putative
kink-turn motif. We functionally characterized the
26 SECIS elements encoded in the human genome.
Surprisingly, the SECIS elements displayed a wide
range of UGA recoding activities, spanning several
1000-fold in vivo and several 100-fold in vitro. The
difference in activity between a representative
strong and weak SECIS element was not explained
by differential binding affinity of SECIS binding
Protein 2, a limiting factor for selenocysteine
incorporation. Using chimeric SECIS molecules, we
identified the internal loop and helix 2, which flank
the kink-turn motif, as critical determinants of UGA
recoding activity. The simultaneous presence of a
GC base pair in helix 2 and a U in the 5’-side of the
internal loop was a statistically significant predictor
of weak recoding activity. Thus, the SECIS contains
intrinsic information that modulates selenocysteine
Selenium is an essential trace element for human health.
Epidemiological studies report that selenium protects
degenerative disorders, reduces the progression of HIV
in infected patients and diminishes mortality in the aging
population (1–4). Most of the beneficial effects of selenium
are likely due to its presence as selenocysteine in a specific
group of proteins, called selenoproteins. Extensive com-
puter analyses of the human genome identified what is
now thought to be the complete set of selenoprotein
genes (5). The human selenoproteome is composed of at
least 25 selenoproteins, although some genes may lead to
the production of more than one isoform (6). Many
human selenoproteins are enzymes involved in oxido-
reduction reactions. Enzymatic activities have been
characterized for approximately one half of the seleno-
iodothyronine deiodinases (Dio1–Dio3), methionine sulf-
oxide reductase (MsrB1 or SelX) and selenophosphate
synthetase (Sps2). In many cases, the selenocysteine resi-
due plays a vital role for enzymatic activity, given its
known or predicted location in the catalytic site (7–9).
However, the remaining human selenoproteins have
not been assigned a precise function due to their recent
bioinformatic identification in the genome.
Selenocysteine is considered as the 21st amino acid,
since its incorporation into the protein sequence depends
on the translational recoding of an in-frame UGA codon
as reviewed in (10–14). Eukaryotes have evolved an intri-
cate strategy to use the UGA as a sense codon for seleno-
cysteine in selenoprotein messenger RNAs (mRNAs),
while maintaining its use as a stop codon in other cellular
mRNAs. The 30-untranslated region (30-UTR) of mamma-
lian selenoprotein mRNAs always harbor a selenocysteine
insertion sequence (SECIS) downstream of the UGA
codon, which is essential for selenocysteine incorporation.
Trans-acting recoding factors identified during the past
decade include a selenocysteine tRNASec(Sec-tRNASec)
(15,16), a dedicated elongation factor (EFsec) (17,18),
and two proteins that bind to the SECIS element:
SECIS binding protein 2 (SBP2) (19,20) and ribosomal
protein L30 (21). It has been proposed that SBP2, which
is limiting in cells, functions as a discriminatory factor and
*To whom correspondence should be addressed. Tel: (33) 1 69 82 32 13; Fax: (33) 1 69 82 31 40; Email: email@example.com
? 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
dictates the expression of the selenoproteome (20–23). The
mechanism of selenocysteine incorporation, particularly
the sequence of events and the nature of the complexes
leading to UGA recoding, is not fully understood yet and
is discussed in (10–14).
Eukaryotic SECIS elements are not highly conserved
at the nucleotide level, but their secondary structures
can be represented as a consensus stem-loop–stem-loop
folding, as illustrated in Figure 1A. Two conserved
motifs in the SECIS are essential for faithful recoding of
UGA as selenocysteine: the SECIS core that contains a
quartet of non-Watson–Crick base pairs and an AAA/G
motif (24–28). The precise distance between these two
regions ismaintained by
(Figure 1A). The SECIS core encompasses two sheared
tandem G–A pairs (hereafter referred to as tandem GA
pairs), which are conserved in all eukaryotic SECIS
elements (29) and are required for the binding of SBP2
and L30 (19–21). In other RNAs with a known 3D
structure, the tandem GA pairs are characteristic of a
kink-turn motif, which can adopt distinct conformations
in solution (30,31). We proposed that the putative kink-
turn motif in the SECIS allows the element to act as a
molecular switch by undergoing dynamic conformational
changes in response to protein binding (21). In contrast to
the SECIS core, the essential AAA/G motif has no known
function or interacting partner. This motif is replaced by
CCX in some SECIS elements (5).
Although it is well-established that the SECIS core and
AAA/G motif are required for selenoprotein synthesis,
little is known about the determinants in SECIS elements
from endogenous selenoprotein mRNAs that might regu-
late the efficiency of selenocysteine incorporation in vivo.
The region surrounding the tandem GA pairs, which is
highly variable among SECIS elements, could affect the
kink-turn folding of the RNA and/or the binding activities
of SBP2 and L30. In addition to the SECIS, the efficiency
of UGA recoding is affected by other cis-acting elements
in the selenoprotein mRNA, including (i) the seleno-
cysteine codon context (32), (ii) the distance between
UGA codon and the SECIS (26,33) and (iii) the presence
of a stem-loop structure adjacent to the UGA codon in a
subset of selenoprotein mRNAs (34,35). However, in all
these cases, the modulation of UGA recoding efficiency
did not exceed a 10- to 20-fold difference.
The purpose of this work is to investigate a potential
regulatory effect of the SECIS on selenoprotein expression
on the scale of the human selenoproteome. We analyzed
the UGA recoding efficiencies of the 26 human SECIS
elements in transfected cells and in an in vitro translation
system. Unexpectedly, we found that SECIS elements
vary dramatically in their ability to direct UGA recoding,
with the differences ranging 4900- and 200-fold in cells
and in vitro, respectively. Using chimeric SECIS elements,
we showed that the internal loop and helix 2 contain
major determinants that control the strength of UGA
recoding. Sequence conservation analysis of the regions
surrounding the kink-turn motif in human SECIS
elements identified statistical predictors of weak UGA
abase paired structure
MATERIALS AND METHODS
Coding sequence of firefly luciferase (Photinus pyralis)
was previously cloned in pcDNA3.1 vector (Invitrogen).
PacI and NotI restriction sites are present immediately
downstream of the UAA stop codon. Additionally, the
luciferase sequence was modified at position 258 to con-
tain an in frame UGA codon. This construct has been
designed to quantitatively analyze the influence of any
SECIS element on UGA recoding efficiency in vivo and
in vitro as described in (21,33,36). The 26 SECIS element
sequences were found in the human genome database and
used to design primers for RT-PCR amplification and
cloning. To have SECIS elements of similar size that
would encompass the minimum active domain for all
selenoprotein mRNAs, we selected 103-nt long sequences
starting ?30bp before the highly conserved ATGA motif
(as listed in Figure 1B). PCR primers were designed to
contain either a PacI or NotI restriction site (listed in
Supplementary Figure S1), in the forward and reverse
primers, respectively, for subcloning into pcDNA3.1
luciferase UGA258vector. Total RNAs were extracted
from Hek293 and HepG2 cells using TRIzol reagent
(Invitrogen) following the manufacturer’s instructions.
Total RNAs (5mg) were annealed with 50pmol of
M-MLV retrotranscriptase (Promega) in 40ml reaction.
Two microliter of this reaction was used for the PCR
amplification using Platinum Pfx DNA polymerase
(Invitrogen) and 4mM of adequate primers. PCR products
and pcDNA3.1 luciferase UGA258vector were digested
with PacI and NotI enzymes, purified and ligated with
T4 DNA Ligase after dephosphorylation of the vector.
Quick change site-directed mutagenesis (adapted from
and UGU258) mutants
oligonucleotides containing the designed mutations (see
Supplementary Figure S2) and the PfuTurbo DNA poly-
merase (Stratagene). To generate the SECIS chimeric
constructs of SelX and Gpx3 listed in Supplementary
Figure S3, we used pairs of overlapping oligonucleotides
with a 30-overhang that were completed with Pwo
DNA polymerase. PCR amplifications were performed
using 4mM of either the set of Gpx3 or SelX SECIS
primers, designed to add PacI and NotI restriction sites.
For RNA electromobility shift assay (REMSA) experi-
ments, luciferase UGA258-Gpx3 and luciferase UGA258-
SelX constructs were digested with PacI and NotI
enzyme to clone the SECIS element downstream of a T7
RNA polymerase promoter in a pUC19 vector. All the
construct sequences were verified by automated DNA
Cell culture and transfection
Hek293 (Invitrogen) and HepG2 (ATTC) cells were grown
and maintained in 100mm plates in Dulbecco’s Modified
Eagle Medium (D-MEM) and in minimum essential
medium (MEM) supplemented with non-essential amino
Nucleic Acids Research, 2009,Vol.37, No. 175869
acids, respectively. Media were supplemented with 10%
fetal bovine serum, 100mg/ml streptomycin, 100UI/ml
penicillin, 1mM sodium pyruvate and 2mM L-glutamine.
Media and supplements were purchased from Invitrogen.
Cells were cultivated in 5% CO2at 378C and humidified
performed to transiently transfect the cells with 6mg of
plasmid (5mg of luciferase and 1mg of b-galactosidase
vectors). Cells were split the day prior to transfection.
Media was changed 24-h post-transfection and cells
collected at 48h with 300ml of lysis buffer (25mM Tris–
phosphate pH 7.8, 2mM DTT, 2mM EDTA, 1% Triton
X-100, 10% glycerol). Cell extracts were assayed for
luciferase and b-galactosidase activities by chemilumines-
cence (Promega Luciferase and Beta-Glo assay systems,
respectively), in triplicate using a microplate reader
FLUOstar OPTIMA (BMG Labtech).
In vitro transcription and translation
Luciferase-SECIS pcDNA3.1 plasmids were linearized
by NotI enzyme and used as templates for in vitro tran-
scription using T7 RNA polymerase (Ribomax kit,
Promega) according to the manufacturer’s instructions.
Cap analog was added to the reaction at a 4:1 ratio
(versus GTP) to generate 50-capped mRNAs. In vitro
translation reactions were assembled in a total volume
of 12.5ml that includes 100ng of luciferase mRNA
(0.19pmol), 50ng of recombinant SBP2 (amino acids
399–846), 8.25ml of nuclease-treated rabbit reticulocyte
lysate (RRL) (Promega), complete amino acid mixture
and RNA guard as described in (37). After 30min
incubation at 378C, translation reactions were assayed in
triplicate for luciferase activity as described earlier.
RNA probes synthesis, electrophoretic mobility
shift assays and competitions
The SECIS probes were synthesized in vitro from
linearized templates with T7 RNA polymerase using
1mM GTP, 1mM ATP, 1mM CTP and 1mM UTP and
10mCi of32P-labeled UTP for 3h at 378C. The transcrip-
tion reactions were treated with DNase RQ1 for 15min
at 378C and then phenol:chloroform extracted. The aque-
ous phase was passed through a Micro Bio-Spin column
according to the manufacturer’s instructions (Bio-Rad).
The REMSA binding buffer includes 30mM Tris, pH
7.8, 8% glycerol, 75mM KCl, 1mM EDTA, 0.25mg/ml
BSA and 0.20mg/ml tRNA. The
RNAs (10fmol) were incubated with the indicated
amount of recombinant SBP2-RBD (amino acids 517–
777), which was expressed in bacteria and purified as
described in (36). After incubation at 378C for 10min,
the complexes were resolved on 8% nondenaturing poly-
acrylamide gels as described in (21). The dried gels
were exposed to autoradiographic screens, which were
analyzed by a Typhoon 9400 scanner (GE Healthcare).
Quantitation of radioactive species was performed with
ImageQuant software (Molecular Dynamics).
The 26 SECIS elements found in the human genome
were selected and cloned in a luciferase-based
Previous studies that analyzed the efficiency of UGA
recoding tested only SECIS elements from a subset of
selenoprotein mRNAs or used SECIS elements of dissimi-
lar lengths (23,38,39). The human selenoproteome is
composed of 25 selenoproteins that contain a single
selenocysteine residue, with the exception of selenoprotein
P (SelP), which has 10 selenocysteines.
selenoprotein, a SECIS element has been identified in
the 30-UTR. In the case of SelP, two functional SECIS
have been characterized (thereafter referred as SelP1 and
SelP2). It has been proposed that SelP2 recodes the initial
UGA of SelP mRNA, while SelP1 is dedicated to the
processive recoding of the downstream UGA triplets
(40). To gain comprehensive insight into the intrinsic in-
fluence of the SECIS element on UGA recoding efficiency,
we cloned the 26 human SECIS elements listed in
Figure 1B. All the sequences were 103nt in length, starting
?30nt before the highly conserved AUGA motif, which
should encompass the minimum active domain (24–28).
As illustrated in Figure 1A, the human SECIS elements
all contain a four-motif secondary structure (stem-loop–
stem-loop) despite the poor primary sequence conserva-
tion. To direct faithful UGA recoding, the SECIS requires
the highly conserved nucleotides shown in the schematic
Figure 1A and represented in bold in Figure 1B. These
include the 50-AUGA and 30-GA at the base of helix 2
and the AAA/G motif (or CCX) in the apical region.
The AUGA and GA motifs close the internal loop by
forming the tandem GA pairs that resemble an RNA
kink-turn motif. As shown in Figure 1A, the AAA/G
(or CCX) motif is found either in a loop or in a bulge in
the apical region of the SECIS element. This difference has
been used to classify the SECIS elements as a Type 1
(loop) or Type 2 (bulge) SECIS, respectively (25,28,41).
Another conserved feature is the length of helix 2 (14–15
base pairs) that ensures a precise distance between the
AUGA and AAA/G (or CCX) motifs. The internal loop
is closed by helix 1, which is variable in size and compos-
ition but important for UGA recoding (27).
Human SECIS elements display a wide range of UGA
recoding activities in vivo and in vitro
To analyze UGA recoding efficiency, the 26 human SECIS
elements were cloned downstream of a luciferase coding
sequence, which has been modified to contain an in frame
UGA codon at position 258 (luciferase UGA258-SECIS
constructs). With this construct previously used in
(21,33,36), an active luciferase is made only when the
UGA is recoded as a selenocysteine codon. This reporter
construct has been validated for selenocysteine incorpor-
ation in transfected cells and in an in vitro translation
system. Plasmids containing the luciferase UGA258-
SECIS reporter constructs were transiently transfected in
human kidney (Hek293) and liver (HepG2) cell lines.
To analyze data from three independent transfections,
Nucleic Acids Research, 2009, Vol. 37,No. 17
we arbitrarily expressed the luciferase activities relative to
the activity measured for SelX, which was expressed as
100%. The results obtained in Hek293 and HepG2 cell
lines showed that, in contrast to previous studies, the
nature of the SECIS element had a dramatic influence
on the efficiency of UGA recoding in vivo (Figure 2A
and B). We observed an ?2250-fold difference between
the strongest (SelN) and the weakest (Gpx6) constructs in
Hek293 cells (Figure 2A). The amplitude is even larger in
HepG2 cells with an ?4900-fold difference between
the reporters containing the Dio3 and SelS SECIS
elements (Figure 2B). Moreover, in both cell lines, we
Figure 1. SECIS elements from human selenoproteome. (A) Schematic of eukaryotic SECIS elements. Human type 1 elements include Gpx1, Gpx2,
SelN, Dio1 and SelV, whereas the other elements are type 2, containing an additional bulge in the apical loop. The four main structural motifs are
identified by arrows. A bracket indicates the position of the SECIS core that contains a quartet of non-Watson–Crick base pairs. Highly conserved
nucleotides are represented in letters. The dots symbolize the two sheared tandem GA base pairs that are essential for kink-turn structures. The
asterisks indicate the variations from the consensus sequence (AA) that are present as CC in SelM, SelO and Trxr3 SECIS elements. (B) Sequence
alignment of the 26 human SECIS elements that were cloned downstream of luciferase UGA258coding sequence and used in the reporter constructs.
They were obtained from SelenoDB website (51). Highly conserved nucleotides are represented in bold. Nucleotides involved in helix 2 are
underlined. Gpx, glutathione peroxidase; Trxr, thioredoxin reductase; Dio, iodothyronine deiodinase; Sel, selenoprotein; Sel15, 15kDa selenoprotein,
Sps2, selenophosphate synthetase.
Nucleic Acids Research, 2009,Vol.37, No. 175871
Figure 2. The UGA-selenocysteine recoding efficiency is strongly influenced by the nature of the SECIS element in vivo and in vitro. Luciferase
UGA258-SECIS reporter gene constructs were generated with all human SECIS elements and tested in transiently transfected Hek293 (A) or HepG2
(B) cells (black bars), or in the cell free translation system (C, white bars). For in vivo experiments (A and B), each luciferase plasmid was transiently
co-transfected with b-galactosidase plasmid. Enzymatic activities were measured 48-h post-transfection on cell protein extracts. Transfection
efficiencies were normalized by calculating the ratio between luciferase relative to b-galactosidase activities. For in vitro experiments (C), the luciferase
UGA258-SECIS synthetic mRNAs were translated using RRL as described in ‘Materials and Methods’ section. To analyze data from three
independent experiments in vivo and in vitro, the UGA recoding efficiencies were arbitrarily expressed relative to the activity from the luciferase
UGA258-SelX construct, which was set as 100%. The group of constructs that show weak efficiencies (SelH, SelO, SelS, Gpx3, Gpx6 and Trxr3) are
represented at a magnified scale in left panel. (D) Comparison of UGA–selenocysteine recoding efficiencies of our luciferase UGA258-SECIS reporter
gene constructs between the different experimental conditions: HepG2 versus Hek293 transfection (left panel), in vitro translation versus Hek293
tranfection (middle panel) and in vitro translation versus HepG2 tranfection (left panel). The SECIS elements are represented as a function of
recoding efficiency group according to Figure 6: blue circles (weak), red square (moderate) and yellow triangles (strong).
Nucleic Acids Research, 2009, Vol. 37,No. 17
identified a group of six weak SECIS elements that
includes SelS, SelO, SelH, Gpx3, Gpx6 and Trxr3
(Figure 2A and B, left panels). The remaining 20 luciferase
UGA258-SECIS constructs were in the moderate and
strong categories of UGA recoding efficiency, with a 16-
and 30-fold range observed in Hek293 and HepG2 cells,
respectively (Figure 2A and B, right panels). As shown
in Figure 2, the difference between the highest of the
weak elements (SelH) and the lowest of the moderate
elements (Txr1) was 2- to 3-fold in both Hek293 and
HepG2 cells. Importantly, the ranking between the
constructs is very similar between the two cell lines used
in this study as illustrated in Figure 2D (left panel) on a
logarithmic scale. The only movements that occurred were
within the moderate and strong categories, as observed for
SelN and Dio1.
To verify that the different SECIS elements drive UGA
recoding with varying strengths, capped luciferase report-
er construct RNAs were translated in a cell-free in vitro
system using RRL. Aliquots of the translation products
were analyzed for luciferase activity, which was expressed
relative to the activity from the reporter containing the
SelX SECIS. Similar to the results in transfected cells, a
wide variation in UGA recoding efficiencies was observed
in the in vitro assay, although with a smaller range
(Figure 2C). Indeed the strongest SECIS (SelP1) was
?200-fold more efficient in recoding UGA than the
weakest (SelO). The lower amplitude of UGA recoding
efficiencies observed in RRL was likely due to the
unnatural non-competitive environment, the translation
reactions being primed with a single mRNA species. In
transfected cells, the luciferase reporter mRNA containing
the SECIS element would compete with endogenous
selenocysteine insertion machinery, which is likely to mag-
nify the differences observed in vitro. Strikingly, despite
the lower amplitude, the same group of six SECIS
elements that displayed weak UGA recoding activity in
cells also gave low levels of luciferase activity in the
in vitro translation assay. As for the transfected cells, an
almost 3-fold difference was observed between Gpx3, the
highest of the weak elements and Trxr2, the lowest of the
moderate elements (Figure 2C). The moderate and strong
elements were spread out over a 5-fold range (right panel
of Figure 2C). The classification of SECIS elements into
three groups (weak, moderate and strong) is best
illustrated when the data are plotted on a logarithmic
scale (Figure 2D). Our results suggest that the differences
properties of the SECIS. Taken together, our data indicate
that distinct properties of the different SECIS elements
may modulate the efficiency of UGA recoding both
in vivo and in vitro.
Gpx3 and SelX as representative weak and strong SECIS
For the subsequent studies, we chose to focus on Gpx3
and SelX as representative weak and strong SECIS
elements, respectively, since they share common fea-
tures despite their differences in their ability to direct
UGA recoding. In humans, Gpx3 and SelX mRNAs pro-
duce detectable amounts of selenoproteins, with Gpx3
being the most abundant selenoprotein in the blood,
while SelX is found in many cell lines (6). Both elements
are predicted to adopt a type 2 structure (Figure 3A), with
major differences between the Gpx3 and SelX SECIS
elements occurring in the size and composition of the
internal loop. The other structural features of the two
elements differ only in their nucleotide composition but
are similar in size.
We first confirmed that the luciferase activity of the
Gpx3and SelX SECIScontructs was due to UGA recoding
and not due to random stop codon readthrough. We
generated two sets of mutants, the first one disabling essen-
tial motifs from the SECIS, either the tandem GA pairs
(?AUGA mutant of luciferase UGA258-SECIS constructs)
or the AAA/G motif (?AAAC and ?AAAG for SelX and
Gpx3, respectively), the other, replacing the UGA at pos-
ition 258 of the luciferase coding sequence by the UAA
stop codon, which does not support selenocysteine
incorporation. These mutant constructs were analyzed in
transient transfection experiments and in vitro translation
assays (Figure 3B). As previously shown in Figure 2, the
SelX SECIS is 350- and 8-fold more efficient in UGA
recoding than Gpx3 in transfected Hek293 cells and in
the in vitro translation assays, respectively. In every case,
deletion of the AUGA or the AAA/G motif led to a
dramatic decrease in luciferase activity (Figure 3B).
Furthermore, the luciferase UAA258-SECIS mutants
showed a complete loss of luciferase activity, thus
confirming that our in vitro and in vivo assays were specific
for selenocysteine insertion even in the case of the Gpx3
construct that contains a very weak SECIS element. We
have tested mutants in which UGA258was changed to a
UGU cysteine codon. As shown in Figure 3B, the
luciferase UGU258-SECIS constructs gave similar activities
for both the Gpx3 or SelX elements in transfected Hek293
cells and the in vitro translation assay (Figure 3B). This
result excluded the possibility that the weak Gpx3 SECIS
interfered with normal protein synthesis and also indicated
that the Gpx3 SECIS construct did not contain any
mRNA instability elements.
SBP2 binds the Gpx3 and SelX SECIS elements with
The simplest explanation for the wide range of UGA
recoding efficiencies would be that various SECIS
elements are bound with different affinities by SBP2,
which is a limiting factor for selenocysteine incorporation
(20–23). To test this hypothesis, we performed REMSA
analyses using radiolabeled Gpx3 and SelX SECIS
elements (Figure 4A and B, respectively). As a source
of protein, we used increasing amounts of the purified
RNA-binding domain of SBP2 (SBP2-RBD, amino acids
517–777), which has been shown to exhibit selective
SECIS binding activity (36). The SBP2-SECIS complexes
were separated from free probes on native acrylamide gels
and quantified to determine apparent Kdvalues. As shown
in Figure 4, the SBP2-RBD bound to the SelX and Gpx3
SECIS elements with similar affinities, with Kdvalues of
Nucleic Acids Research, 2009,Vol.37, No. 17 5873
8nM and 14nM, respectively. These very similar affinities
for SBP2 cannot explain the difference in UGA recoding
between Gpx3 and SelX SECIS. Thus, we hypothesized
that other determinants in the SECIS may modulate the
efficiency of selenocysteine incorporation.
The internal loop and helix 2 of the SECIS determine
UGA recoding efficiency
To define the motifs in the SECIS that modulate the effi-
ciency of UGA recoding, we performed structure–function
UGA recoding efficiency
UGA recoding efficiency
120 100 806040 200
0210 1008060 40 20
0 0.10.2 0.3
000210 10000 8000 60004000 2000
010000 800060004000 2000
Figure 3. The UGA recoding efficiencies of the SelX and Gpx3 SECIS elements are specific for selenocysteine insertion in vivo and in vitro. (A) The
secondary structures of SelX and Gpx3 SECIS elements are represented in the frame of type 2 category of elements. Consensus motifs are shown in
bold. The A–U, G–C, G–U and noncanonical G–A base pairs are represented by dots. The main four structural motifs are separated by gray bars.
(B) Mutant luciferase-SelX and luciferase-Gpx3 constructs were assayed for UGA recoding activity in transiently transfected Hek293 cells (black
bars) or in the rabbit reticulocyte cell-free translation system (white bars). Mutations targeted either essential motifs the SECIS elements (?AUGA,
?AAAC and ?AAAG) or codon 258 in the luciferase coding sequence (UAA258and UGU258). UGA recoding activities were expressed relative to
the activity of the wild-type luciferase UGA258-SelX construct (set as 100%).
Nucleic Acids Research, 2009, Vol. 37,No. 17
analyses of the SelX and Gpx3 SECIS elements using a
chimera approach (see Supplementary Figure S3 for
sequences, structures and nomenclature). Using this
strategy, we were able to swap individual domains in the
SelX SECIS with the corresponding regions from Gpx3
(Figure 5A and B); convert the weak Gpx3 SECIS into a
stronger element (Figure 5C and D); and systematically
transform the strong SelX SECIS into a weak one
(Figure 5E and F). The chimeric SECIS elements were
cloned downstream of luciferase UGA258coding region.
The constructs were tested in transiently transfected
Hek293 cells (Figure 5, left panels) and in the in vitro
translation assay (Figure 5, right panels).
The first set of experiments (Figure 5A and B) consisted
of replacing domains in the SelX SECIS (shown in black)
with their Gpx3 counterparts (shown in gray). A change in
luciferase activity should be detected when the domains
contained important features for UGA recoding efficiency.
Replacement of helix 1 in SelX with the Gpx3 counterpart
(chimera 1) had very little effect both in vivo and in vitro
(Figure 5A and B). Thus, helix 1 may only be important
for closing the bottom of the internal loop (see the sec-
ondary structure of the SECIS in Figure 3A) as previously
suggested. The most dramatic effects were observed with
the individual swapping of the internal loop (chimera 7),
helix 2 (chimera 12) or both domains (chimera 10), which
decreased UGA recoding efficiency >300-fold, similar to
the level observed for the Gpx3 parental construct. Our
data also clearly indicate that helix 2, which was previous-
ly expected to only physically separate the AUGA from
the AAA/G, harbors determinant(s) for selenocysteine
insertion efficiency. Although the length of helix 2 is simi-
lar in the two SECIS elements, this region is completely
base paired in the SelX SECIS while it contains two A–C
mismatches in Gpx3 that can form non-WC base pairs.
Finally, we analyzed the importance of the essential
AAA/G motif, which is located in a bulge in the apical
loop region of the SelX and Gpx3 SECIS elements. In
contrast to what might be expected for a domain
containing an essential motif, the insertion of a weak
apical loop into the strong SECIS context (chimera 4)
led to only a modest 2- to 5-fold decrease in luciferase
activity. Thus, this region contains minor determinants
that regulate UGA recoding efficiency.
In a second set of experiments, when the motifs from
the strong element were inserted in the weak SECIS
context, very few effects were observed (Figure 5C and
D). Even the swapping of the internal loop induced
little, if any increase in recoding efficiency (chimera 8).
These results suggested that the determinants for the
weak efficiency of Gpx3 are dominant over the strong
determinants in the SelX SECIS. However, the simultan-
eous insertion of the SelX internal loop and helix 2 into
the Gpx3 SECIS (chimera 9) resulted in almost full recov-
ery of UGA recoding activity in cells (Figure 5C) and
in vitro (Figure 5D). Taken together, our data indicate
that the internal loop and helix 2, which both flank the
tandem GA pairs, are necessary and sufficient to switch a
weak SECIS element into a strong one, and vice versa. To
verify this finding, we gradually transformed the strong
SelX SECIS into a weak element from bottom to top
and top to bottom. As shown in Figure 5E and F, the
largest effects were observed when changes occurred in
the internal loop and the helix 2 (chimeras 2 and 3). It is
also clear from these results that negative determinants
won over the positive determinants, as previously
observed with the swapping of individual domains.
Identification of nucleotide sequences that predict weak
UGA recoding activity
To identify nucleotide sequences that may modulate UGA
recoding efficiency, we first analyzed the sequence conser-
vation of the internal loop and helix 2 in the 26 human
SECIS elements using Weblogo (42) (Figure 6). In add-
ition to the previously identified highly conserved motifs
(i.e. ATGA, AAA/G and GA), we noticed that other
positions with lower conservation were present near the
tandem GA pairs in the SECIS core. More precisely, a
Kd (SelX): 8 nM
Kd (Gpx3): 14 nM
Figure 4. SBP2 binds with similar affinity to the SelX (A) and Gpx3
(B) SECIS elements. The
with increasing concentration of the RNA binding domain of SBP2
(SBP2-RBD). The RNA–protein complexes were analyzed on a native
8% polyacrylamide gel. The apparent Kdvalues were determined by
plotting the fraction of protein–SECIS complex as a function of protein
32P-labeled SECIS elements were incubated
Nucleic Acids Research, 2009,Vol.37, No. 175875
potential GC base pair in the lower half of helix 2 is pre-
sent in 62% of the human SECIS elements (illustrated by
a sharp in Figure 6B and indicated as position +2 in
Figure 6C). Interestingly, this GC base pair was found
at the same distance from the tandem GA pairs in many
of the synthetic RNAs selected for in vitro binding to
SBP2 using SELEX methodology (43). Thus, the GC is
an important but not essential RNA feature for SBP2
recognition. Furthermore, the genomic sequences of
65% of human SECIS elements contain a T, 3nt before
the ATGA on the 50-side of the internal loop (illustrated
by an asterisk in Figure 6B and indicated as position –7
UGA recoding efficiency
UGA recoding efficiency
UGA recoding efficiency
UGA recoding efficiency
UGA recoding efficiency
UGA recoding efficiency
Figure 5. Structure-function analyses of SelX and Gpx3 SECIS elements. SelX and Gpx3 were used as a representative weak and strong element,
respectively. Chimeric constructs of these elements were generated based on the four domains shown in Figure 3A and cloned downstream of
luciferase UGA258coding sequence. The composition and nomenclature of the chimeric SECIS elements are represented beneath histograms, using
gray and black to indicate domains originating from Gpx3 and SelX SECIS elements, respectively. The histograms represent data from transiently
transfected Hek293 cells (black bars) or from cell free translation assays (white bars), which were performed as described in the Figure 2 caption. The
UGA recoding efficiencies are expressed relative to the luciferase UGA258-SelX construct (set as 100%). (A and B) Domain swapping of Gpx3 SECIS
domains into the strong SelX element context. (C and D) Domain swapping of SelX SECIS domains into the weak Gpx3 context. (E and F) Gradual
transformation of a strong SECIS element into a weak one, and vice versa.
Nucleic Acids Research, 2009, Vol. 37,No. 17
of the left helix in Figure 6C). Both the GC and the T
nucleotides have also been pointed out as being conserved
features in a recent report, which aligned 286 SECIS
elements from various eukaryote species (29).
We then investigated whether these conserved motifs
are functionally relevant by generating the sequence
logos separately with the weak, moderate or strong
categories of elements (Figure 6D–F). Interestingly, we
observed that the GC and T motifs are over-represented
in weak SECIS elements versus the strong ones. As
shown in Figure 6B, the simultaneous presence of the T
and the GC was found in 100% of weak SECIS elements,
compared to only 25 and 12.5% for the moderate and
strong classes, respectively. The association of both
motifs was a statistically significant predictor of the
weak versus the strong/moderate class of SECIS element
(Fisher test, P<0.002).
The SECIS element, which is essential for selenocysteine
insertion, is found in all selenoprotein mRNAs that
comprise the mammalian selenoproteome. However, the
primary sequence conservation is poor and the four-motif
secondary structure of the SECIS is subject to variability,
notably in the internal and apical loops as illustrated in
Figures 1 and 6. While previous studies reported signifi-
cant differences among SECIS elements in their ability to
drive UGA recoding, these analyses tested only specific
subsets of selenoprotein mRNAs, i.e. Gpx1, Gpx4,
Trxr1, Dio1 and SelP1 in (23) or Gpx1, Gpx2 and Gpx4
in (38,39) and did not identify the determinants in the
SECIS that were responsible for this variability. In this
work, we report the first comprehensive analysis of the
26 SECIS elements encoded in the human genome. We
found that human SECIS elements displayed a wide
range of UGA recoding activities in vivo, spanning several
1000-fold in transfected Hek293 and HepG2 cells and sev-
eral 100-fold in vitro (Figure 2), which is much broader
than that has been previously observed. Surprisingly, this
variability was likely not due to variation in SBP2 binding
activity, an essential and limiting factor for selenocysteine
incorporation, since we found that the weak Gpx3 and
strong SelX SECIS elements were bound with similar
All human SECIS
Figure 6. Sequence alignment of the regions surrounding the kink-turn motif in human SECIS elements as a function of UGA recoding efficiency.
(A) Schematic representation of the consensus SECIS element structure. The left (L) and right (R) sides of helix 2 are numbered in blue relative to
the AUGA and GA nucleotides, respectively. (B) DNA sequences that were used for sequence analyses started 6nt before the ATGA (position-10)
and ended 6nt after the GA motif (position-8). The classification SECIS elements were classified having as weak, moderate and strong UGA
recoding activity based on in vitro experiments (Figure 2C). Highly and moderately conserved nucleotides are boxed in black or gray, respectively.
Consensus nucleotides are represented underneath the sequence alignment. Additional conserved nucleotides are indicated by a sharp and an asterisk,
for the GC base pair and the T, respectively. Sequence logos were generated using Web logo software (42) with all human SECIS (C) or the group of
weak (D), moderate (E) or strong (F) elements. The left part of helix 2 is represented in 50- to 30-orientation above the right counterpart represented
in 30- to 50-orientation to highlight possible consensus base pairing. The overall height of the stack indicates the sequence conservation at each
position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.
Nucleic Acids Research, 2009,Vol.37, No. 17 5877
affinity by SBP2. The consistency of the results is striking
as the SECIS elements were classified almost identically as
weak, moderate or strong by all three experimental
approaches (Figure 2D). Taken together, our results
strongly support the hypothesis that the SECIS element
contains intrinsic information that modulates seleno-
cysteine incorporation efficiency in an SBP2-independent
identifying the nucleotides or structures in an individual
SECIS element that are essential for selenocysteine
incorporation. To pinpoint the RNA structural domain(s)
that define the UGA recoding efficiency of a SECIS, we
generated chimeric molecules using SelX and Gpx3 as a
representative strong and weak element, respectively. Our
data indicate that helix 1 and the apical loop are the minor
determinants for UGA recoding efficiency (Figure 5)
although those regions are essential for the insertion of
selenocysteine (24–28,44). Instead we found that the
internal loop, which varies in composition and size
among SECIS elements, was one important determinant
in Gpx3 and SelX, respectively. Although both SECIS
elements contain internal loops of comparable size (8
and 9nt), they differ in their predicted structures, with
an almost symmetric (4+5) loop in SelX and an asym-
metric (1+7) loop in Gpx3. In our constructs, this
domain was necessary and sufficient to transform a
strong SECIS into a weak one. The converse experiment
of inserting the SelX internal loop into the Gpx3 context
did not rescue the weak element, indicating that more than
one region is involved. Indeed, we found that helix 2,
which was initially thought to act as a spacer to ensure a
precise distance between the tandem GA pairs and the
AAA/G (or CCX) motif, also modulated UGA recoding
activity. Interestingly, the helices are of similar length
(14nt) in these two SECIS elements. However, the SelX
helix 2 is fully base paired, whereas Gpx3 is predicted to
contain two C–A mismatches in this region (Figure 3A).
Finally, we showed that these two RNA structural
domains, i.e. the internal loop and helix 2, together are
necessary and sufficient to regulate the efficiency of UGA
recoding in vivo and in vitro (Figure 5, chimeras 9 and 10).
We also found that the simultaneous presence of a GC
base pair in helix 2 and a T 30-nucleotides before the
ATGA is a statistically significant predictor of weak
UGA recoding activity. Although the two motifs may
not be sufficient to transform a weak into a strong element
or vice versa, they may contribute to selenocysteine
incorporation efficiency by influencing the function of
the adjacent tandem GA pairs, which are essential for
proposed that the tandem GA pairs in the SECIS core
may represent a non-canonical kink-turn motif. The
conserved GC and T nucleotides could also influence the
angle or dynamics of the kink-turn folding. Precise struc-
implications of the context of the tandem GA pairs on
the functional activity of the SECIS.
Although SBP2 has been proposed to dictate the
expression of the selenoproteome, the affinity of SBP2
for the Gpx3 and SelX SECIS elements differed by only
2-fold despite their 350-fold difference in directing UGA
recoding in transfected cells. Thus, a wide range of UGA
recoding efficiencies is not solely explained by SBP2 bind-
ing affinity. This observation is rather surprising as the
SBP2 binding site encompasses helix 2 and the internal
loop (45), the regions we identified as the major determin-
ant of the selenocysteine insertion efficiency. Several
studies have found that SBP2 exhibits selective SECIS
binding activity. In vitro analyses using REMSA have
found that SBP2 exhibits selective high affinity binding
activity to Gpx4 and Trxr1 SECIS (36) while in vivo bind-
ing of selenoprotein mRNAs by SBP2 via immunopre-
cipitation of the proteins and quantitation of bound
mRNAs have shown that SBP2 exhibits strong preferen-
tial binding to SelW, Sel15, Gpx4, SelH, Trxr1, Dio2
and SelK mRNAs over others (22). Indeed the difference
in SBP2 binding affinity does explain the preferential syn-
thesis of Gpx4 over Gpx1 and Dio2 in a competitive
in vitro translation assay (36). However, the regulation
of the selenoproteome appears to be more complex as
there is no obvious correlation between the reported bind-
ing properties of SBP2 and the UGA recoding efficiencies
that we observed, suggesting the involvement of additional
cis- or trans-acting factors.
The most surprising finding from our study was the
identification of a small group of SECIS elements,
including Gpx3, Gpx6, SelH, SelO, SelS and Trxr3, that
directed UGA recoding with extremely low efficiency both
in vivo and in vitro. For Gpx3, we used mutations in either
the apical loop or the tandem GA pairs of the SECIS
elements to demonstrate that the low level of luciferase
activity that we detected was due to UGA recoding as
selenocysteine and not to a low level of translational
readthrough as observed for a genuine stop codon
(Figure 3). Metabolic labeling studies demonstrated that
three other weak SECIS elements (SelH, SelO and SelS)
directed selenocysteine incorporation in a reporter con-
struct in transfected cells although no quantitative analysis
was reported in this study (5). These results raise the ques-
tion as to how selenoproteins with an inefficient SECIS
element are made in significant amounts in vivo. One
possibility is that the abundance of a selenoprotein
mRNA may compensate for the weak efficiency of its
SECIS element. Interestingly, Gpx3 is mainly produced
in the kidney and the Gpx3 mRNA is ranked as the
most abundant selenoprotein mRNA in this tissue in
mice (46). However, the other selenoprotein mRNAs
with weak SECIS elements are expressed at low or mod-
erate levels in mouse tissues (46).
The more likely explanation for our results is that the
selenocysteine incorporation efficiency of a weak SECIS
element may be enhanced by other cis-acting elements
within the transcript. Several selenoprotein mRNAs con-
tain RNA structures in the coding region that modulate
UGA recoding efficiency (34,35). However, none of these
structures are present in the selenoproteins mRNAs that
we identified as containing weak SECIS elements. A
largely unexplored area in the field is whether the activity
of a SECIS element is influenced by other sequences in the
30-UTR of the transcript. Previous studies have analyzed
minimal SECIS elements of 50–100nt, which are sufficient
Nucleic Acids Research, 2009, Vol. 37,No. 17
to support UGA recoding in vitro and in cells (19,41,47).
We designed our SECIS elements to encompass ?100nt,
which is the minimal sequence required for binding of
SBP2 and L30 to the Gpx4 SECIS (21,45). For some
selenoprotein mRNAs, additional sequences flanking the
SECIS may be required for efficient selenocysteine
incorporation. Indeed, a very recent study analyzing the
expression of human Gpx3 in transfected cells reported
that selenocysteine incorporation was supported by a
30-UTR of 500nt, but not by a 100nt 30-UTR that
encompassed the SECIS (48). While the underlying basis
for this difference was not investigated, we speculate the
additional sequences may be required to stabilize the stem-
loop structure of the SECIS or to recruit other factors that
may enhance the efficiency of UGA recoding. Finally, one
must consider the possibility that the activity of a weak
SECIS element may be enhanced in vivo by cell-type
specific or selenoprotein-specific trans-acting factors that
are not present in our experimental systems. These may be
proteins, co-factors, subcellular localization of proteins
and mRNAs, or differing oxidation states of UGA
recoding components [as observed for SBP2 in (49)].
Indeed, SBP2 interact with proteins involved in the assem-
bly of ribonucleoprotein complexes, including the R2TP
complex and Nufip, which could influence selenocysteine
incorporation in vivo (50). Such differences may also
account for the wider range of UGA recoding activities
that we observed in vivo, compared with in vitro.
Regardless of the explanation, our results clearly show
that the 26 human SECIS elements contain intrinsic infor-
mation that modulates their ability to direct selenocysteine
incorporation. Our findings have implications not only
for designing experiments to analyze SECIS function but
also for understanding how the expression of the
selenoproteome is regulated in vivo.
Supplementary Data are available at NAR Online.
We are grateful to Claude Thermes and Yves d’Aubenton-
Carafa for helpful discussions on weblogo sequence
Bubenik for providing the SBP2-RBD plasmid and for
critical reading of the manuscript.
analyses andto Jodi
CNRS (ATIP program to L.C.); the Fondation pour la
Recherche Me ´ dicale (to L.C.); the Ligue Contre le Cancer
(Comite ´ de l’Essonne, to L.C.); the Association pour la
Recherche sur le Cancer (grants 4849 to L.C. and 4891 to
O.J.-J.); and the National Institutes of Health (grant
HL29582 to D.M.D.); fellowship from the Ministe ` re de
l’Enseignement Supe ´ rieur et de la Recherche (to L.L.).
Funding for open access charge: CNRS.
Conflict of interest statement. None declared.
1. Rayman,M.P. (2005) Selenium in cancer prevention: a review
of the evidence and mechanism of action. Proc. Nutr. Soc., 64,
2. Patrick,L. (2004) Selenium biochemistry and cancer: a review of the
literature. Altern. Med. Rev., 9, 239–258.
3. Birringer,M., Pilawa,S. and Flohe,L. (2002) Trends in selenium
biochemistry. Nat. Prod. Rep., 19, 693–718.
4. Rayman,M.P. (2000) The importance of selenium to human health.
Lancet, 356, 233–241.
5. Kryukov,G.V., Castellano,S., Novoselov,S.V., Lobanov,A.V.,
Zehtab,O., Guigo,R. and Gladyshev,V.N. (2003) Characterization
of mammalian selenoproteomes. Science, 300, 1439–1443.
6. Fomenko,D.E., Novoselov,S.V., Natarajan,S.K., Lee,B.C., Koc,A.,
Carlson,B.A., Lee,T.H., Kim,H.Y., Hatfield,D.L. and
Gladyshev,V.N. (2009) MsrB1 (methionine-R-sulfoxide reductase 1)
knock-out mice: roles of MsrB1 in redox regulation and
identification of a novel selenoprotein form. J. Biol. Chem., 284,
7. Kim,H.Y., Fomenko,D.E., Yoon,Y.E. and Gladyshev,V.N. (2006)
Catalytic advantages provided by selenocysteine in methionine-
S-sulfoxide reductases. Biochemistry, 45, 13697–13704.
8. Rocher,C., Lalanne,J.L. and Chaudiere,J. (1992) Purification and
properties of a recombinant sulfur analog of murine selenium–
glutathione peroxidase. Eur. J. Biochem., 205, 955–960.
9. Berry,M.J., Maia,A.L., Kieffer,J.D., Harney,J.W. and Larsen,P.R.
(1992) Substitution of cysteine for selenocysteine in type I
iodothyronine deiodinase reduces the catalytic efficiency of the
protein but enhances its translation. Endocrinology, 131, 1848–1852.
10. Berry,M.J., Tujebajeva,R.M., Copeland,P.R., Xu,X.M.,
Carlson,B.A., Martin,G.W. III, Low,S.C., Mansell,J.B.,
Grundner-Culemann,E., Harney,J.W. et al. (2001) Selenocysteine
incorporation directed from the 30UTR: characterization of
eukaryotic EFsec and mechanistic implications. Biofactors, 14,
11. Driscoll,D.M. and Copeland,P.R. (2003) Mechanism and regulation
of selenoprotein synthesis. Annu. Rev. Nutr., 23, 17–40.
12. Hatfield,D.L., Carlson,B.A., Xu,X.M., Mix,H. and Gladyshev,V.N.
(2006) Selenocysteine incorporation machinery and the role of
selenoproteins in development and health. Prog. Nucleic Acid Res.
Mol. Biol., 81, 97–142.
13. Papp,L.V., Lu,J., Holmgren,A. and Khanna,K.K. (2007) From
selenium to selenoproteins: synthesis, identity, and their role in
human health. Antioxid. Redox Signal., 9, 775–806.
14. Allmang,C., Wurth,L. and Krol,A. (2009) The selenium to
selenoprotein pathway in eukaryotes: more molecular partners than
anticipated. Biochim. Biophys. Acta. (in press).
15. Hatfield,D., Lee,B.J., Hampton,L. and Diamond,A.M. (1991)
Selenium induces changes in the selenocysteine tRNA[Ser]Sec
population in mammalian cells. Nucleic Acids Res., 19, 939–943.
16. Lee,B.J., Worland,P.J., Davis,J.N., Stadtman,T.C. and
Hatfield,D.L. (1989) Identification of a selenocysteyl-tRNA(Ser)
in mammalian cells that recognizes the nonsense codon, UGA.
J. Biol. Chem., 264, 9724–9727.
17. Fagegaltier,D., Hubert,N., Yamada,K., Mizutani,T., Carbon,P. and
Krol,A. (2000) Characterization of mSelB, a novel mammalian
elongation factor for selenoprotein translation. EMBO J., 19,
18. Tujebajeva,R.M., Copeland,P.R., Xu,X.M., Carlson,B.A.,
Harney,J.W., Driscoll,D.M., Hatfield,D.L. and Berry,M.J. (2000)
Decoding apparatus for eukaryotic selenocysteine insertion. EMBO
Rep., 1, 158–163.
19. Copeland,P.R. and Driscoll,D.M. (1999) Purification, redox
sensitivity, and RNA binding properties of SECIS-binding protein
2, a protein involved in selenoprotein biosynthesis. J. Biol. Chem.,
20. Copeland,P.R., Fletcher,J.E., Carlson,B.A., Hatfield,D.L. and
Driscoll,D.M. (2000) A novel RNA binding protein, SBP2, is
required for the translation of mammalian selenoprotein mRNAs.
EMBO J., 19, 306–314.
21. Chavatte,L., Brown,B.A. and Driscoll,D.M. (2005) Ribosomal
protein L30 is a component of the UGA-selenocysteine recoding
machinery in eukaryotes. Nat. Struct. Mol. Biol., 12, 408–416.
Nucleic Acids Research, 2009,Vol.37, No. 175879
22. Squires,J.E., Stoytchev,I., Forry,E.P. and Berry,M.J. (2007) SBP2 Download full-text
binding affinity is a major determinant in differential selenoprotein
mRNA translation and sensitivity to nonsense-mediated decay.
Mol. Cell Biol., 27, 7848–7855.
23. Low,S.C., Grundner-Culemann,E., Harney,J.W. and Berry,M.J.
(2000) SECIS-SBP2 interactions dictate selenocysteine incorporation
efficiency and selenoprotein hierarchy. EMBO J., 19, 6882–6890.
24. Berry,M.J., Banu,L., Chen,Y.Y., Mandel,S.J., Kieffer,J.D.,
Harney,J.W. and Larsen,P.R. (1991) Recognition of UGA as a
selenocysteine codon in type I deiodinase requires sequences in the
30untranslated region. Nature, 353, 273–276.
25. Fagegaltier,D., Lescure,A., Walczak,R., Carbon,P. and Krol,A.
(2000) Structural analysis of new local features in SECIS RNA
hairpins. Nucleic Acids Res., 28, 2679–2689.
26. Martin,G.W. III, Harney,J.W. and Berry,M.J. (1996) Selenocysteine
incorporation in eukaryotes: insights into mechanism and efficiency
from sequence, structure, and spacing proximity studies of the type
1 deiodinase SECIS element. RNA, 2, 171–182.
27. Martin,G.W. III, Harney,J.W. and Berry,M.J. (1998) Functionality
of mutations at conserved nucleotides in eukaryotic SECIS elements
is determined by the identity of a single nonconserved nucleotide.
RNA, 4, 65–73.
28. Walczak,R., Westhof,E., Carbon,P. and Krol,A. (1996) A novel
RNA structural motif in the selenocysteine insertion element of
eukaryotic selenoprotein mRNAs. RNA, 2, 367–379.
29. Chapple,C.E., Guigo,R. and Krol,A. (2009) SECISaln, a web-based
tool for the creation of structure-based alignments of eukaryotic
SECIS elements. Bioinformatics, 25, 674–675.
30. Goody,T.A., Melcher,S.E., Norman,D.G. and Lilley,D.M. (2004)
The kink-turn motif in RNA is dimorphic, and metal ion-
dependent. RNA, 10, 254–264.
31. Matsumura,S., Ikawa,Y. and Inoue,T. (2003) Biochemical
characterization of the kink-turn RNA motif. Nucleic Acids Res.,
32. Gupta,M. and Copeland,P.R. (2007) Functional analysis of the
interplay between translation termination, selenocysteine codon
context, and selenocysteine insertion sequence-binding protein 2.
J. Biol. Chem., 282, 36797–36807.
33. Mehta,A., Rebsch,C.M., Kinzy,S.A., Fletcher,J.E. and
Copeland,P.R. (2004) Efficiency of mammalian selenocysteine
incorporation. J. Biol. Chem., 279, 37852–37859.
34. Howard,M.T., Moyle,M.W., Aggarwal,G., Carlson,B.A. and
Anderson,C.B. (2007) A recoding element that stimulates decoding
of UGA codons by Sec tRNA[Ser]Sec. RNA, 13, 912–920.
35. Howard,M.T., Aggarwal,G., Anderson,C.B., Khatri,S.,
Flanigan,K.M. and Atkins,J.F. (2005) Recoding elements located
adjacent to a subset of eukaryal selenocysteine-specifying UGA
codons. EMBO J., 24, 1596–1607.
36. Bubenik,J.L. and Driscoll,D.M. (2007) Altered RNA binding
activity underlies abnormal thyroid hormone metabolism linked to
a mutation in selenocysteine insertion sequence-binding protein 2.
J. Biol. Chem., 282, 34653–34662.
37. Copeland,P.R. and Driscoll,D.M. (2002) Purification and analysis
of selenocysteine insertion sequence-binding protein 2. Methods
Enzymol., 347, 40–49.
38. Muller,C., Wingler,K. and Brigelius-Flohe,R. (2003) 30UTRs of
glutathione peroxidases differentially affect selenium-dependent
mRNA stability and selenocysteine incorporation efficiency. Biol.
Chem., 384, 11–18.
39. Wingler,K., Bocher,M., Flohe,L., Kollmus,H. and
Brigelius-Flohe,R. (1999) mRNA stability and selenocysteine
insertion sequence efficiency rank gastrointestinal glutathione
peroxidase high in the hierarchy of selenoproteins. Eur. J. Biochem.,
40. Stoytcheva,Z., Tujebajeva,R.M., Harney,J.W. and Berry,M.J.
(2006) Efficient incorporation of multiple selenocysteines
involves an inefficient decoding step serving as a potential
translational checkpoint and ribosome bottleneck. Mol. Cell Biol.,
41. Grundner-Culemann,E., Martin,G.W. III, Harney,J.W. and
Berry,M.J. (1999) Two distinct SECIS structures capable of
directing selenocysteine incorporation in eukaryotes. RNA, 5,
42. Crooks,G.E., Hon,G., Chandonia,J.M. and Brenner,S.E. (2004)
WebLogo: a sequence logo generator. Genome Res., 14, 1188–1190.
43. Clery,A., Bourguignon-Igel,V., Allmang,C., Krol,A. and
Branlant,C. (2007) An improved definition of the RNA-binding
specificity of SECIS-binding protein 2, an essential component of
the selenocysteine incorporation machinery. Nucleic Acids Res., 35,
44. Lesoon,A., Mehta,A., Singh,R., Chisolm,G.M. and Driscoll,D.M.
(1997) An RNA-binding protein recognizes a mammalian
selenocysteine insertion sequence element required for
cotranslational incorporation of selenocysteine. Mol. Cell Biol., 17,
45. Fletcher,J.E., Copeland,P.R., Driscoll,D.M. and Krol,A. (2001)
The selenocysteine incorporation machinery: interactions between
the SECIS RNA and the SECIS-binding protein SBP2. RNA, 7,
46. Hoffmann,P.R., Hoge,S.C., Li,P.A., Hoffmann,F.W.,
Hashimoto,A.C. and Berry,M.J. (2007) The selenoproteome exhibits
widely varying, tissue-specific dependence on selenoprotein P for
selenium supply. Nucleic Acids Res., 35, 3963–3973.
47. Shen,Q., McQuilkin,P.A. and Newburger,P.E. (1995) RNA-binding
proteins that specifically recognize the selenocysteine insertion
sequence of human cellular glutathione peroxidase mRNA. J. Biol.
Chem., 270, 30448–30452.
48. Ottaviano,F.G., Tang,S.S., Handy,D.E. and Loscalzo,J. (2009)
Regulation of the extracellular antioxidant selenoprotein plasma
glutathione peroxidase (GPx-3) in mammalian cells. Mol. Cell
Biochem., 327, 111–126.
49. Papp,L.V., Lu,J., Striebel,F., Kennedy,D., Holmgren,A. and
Khanna,K.K. (2006) The redox state of SECIS binding protein 2
controls its localization and selenocysteine incorporation function.
Mol. Cell Biol., 26, 4895–4910.
50. Boulon,S., Marmier-Gourrier,N., Pradet-Balade,B., Wurth,L.,
Verheggen,C., Jady,B.E., Rothe,B., Pescia,C., Robert,M.C., Kiss,T.
et al. (2008) The Hsp90 chaperone controls the biogenesis
of L7Ae RNPs through conserved machinery. J. Cell Biol., 180,
51. Castellano,S., Gladyshev,V.N., Guigo,R. and Berry,M.J. (2008)
SelenoDB 1.0: a database of selenoprotein genes, proteins and
SECIS elements. Nucleic Acids Res., 36, D332–D338.
Nucleic Acids Research, 2009, Vol. 37,No. 17