Biochimica et Biophysica Acta 1830 (2013) 2728–2738

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagen

Translational repression of the McKusick–Kaufman syndrome transcript by unique

upstream open reading frames encoding mitochondrial proteins with alternative
polyadenylation sites
Chizuru Akimoto a, b, 1, Eiji Sakashita b, 1, Katsumi Kasashima b, Kenji Kuroiwa b, Kaoru Tominaga b,
Toshiro Hamamoto b, Hitoshi Endo b,⁎
a
Division of Neurology, Department of Internal Medicine, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan
b
Department of Biochemistry, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background: Upstream open reading frames (uORFs) are commonly found in the 5′-untranslated region (UTR) of
Received 15 September 2012 many genes and function in translational control. However, little is known about the existence of the proteins
Received in revised form 20 November 2012 encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs
Accepted 11 December 2012 of the McKusick–Kaufman syndrome (MKKS) gene that causes a genetic disorder.
Available online 21 December 2012
Methods: Northern blotting, 3′-RACE, and bioinformatics were used for determining the length of transcripts and
their 3′ ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity
Keywords:
Upstream open reading frame
of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein
Polyadenylation signal products and their subcellular localization.
mRNA processing Results: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs
Translational regulation and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the
McKusick–Kaufman syndrome 5′-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream
Mitochondrion ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial mem-
brane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation
sites at the 5′-UTR are atypical but surely exist in human transcripts.
Conclusions: Multiple uORFs at the 5′-UTR of the MKKS long transcript function as translational repressor for
MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane.
General significance: Our findings provide unique insights into production of uORF-derived peptides and functions
of uORFs.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction demonstrated that uORFs are widespread in mammalian transcripts,

The regulation of gene expression is controlled at many levels,
including transcriptionally and during mRNA processing, protein
translation, and protein turnover. Translation is often controlled by
sequence elements in both the 5′- and 3′-untranslated regions
(UTRs) of mRNA. Upstream open reading frames (uORFs) have been
identified as key cis regulatory elements for the initiation of transla-
tion of the main downstream reading frame of a transcript [1]. In
early studies of mammalian genes, uORFs were thought to be present
in b 10% of mRNAs [2]. However, recent genome-wide studies have
Abbreviations: BBS6, Bardet–Biedl syndrome 6; ESTs, expression sequence tags;
IRES, internal ribosome entry site; MKKS, McKusick–Kaufman syndrome; PS,
polyadenylation signal; uORF, upstream open reading frame; UTR, untranslated region;
WT, wild-type; RACE, rapid amplification of cDNA ends
⁎ Corresponding author. Tel.: +81 285 58 7322; fax: +81 285 44 1827.
E-mail address: hendo@jichi.ac.jp (H. Endo).
1
These authors contributed equally to this work.
with approximately half of human transcripts containing at least one
uORF [3,4]. Proteomic analysis of small proteins in human leukemia
K562 cells has revealed the presence of several dozen uORF-derived
peptides in vivo [5], and in some cases, the expression of the uORFs
correlates with genetic polymorphisms [6], stress [7,8], and disease
phenotype [9]. A number of the uORFs correlate with a reduction in
translation of the downstream reading frame [4]. This is because the
initiation codon of the uORF arrests the scanning of the 40S ribosomal
subunit and its initiation factors prior to reaching the downstream
start codon in a polycistronic transcript [10]. In a small number of mam-
malian uORFs, such as ribosomal proteins, a nascent peptide translated
from the uORF directly affects the translation of the downstream read-
ing frame [11–13]. However, there is only limited evidence for the
presence of the uORF-derived peptides in vivo. To bypass the AUG
codon of a uORF and enable protein synthesis of the downstream read-
ing frame, several mechanisms (e.g., leaky scanning, reinitiation, and
cellular internal ribosome entry site (IRES)) have been proposed [14].

0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2012.12.010
 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738
Although the significance of uORFs is well documented in the context
of the regulation of translation of the downstream reading frame,
there is limited evidence of endogenous uORF-derived proteins and
their physiological importance.
McKusick–Kaufman syndrome (MKKS [OMIM: 236700]), also known
as Bardet–Biedl syndrome 6 (BBS6 [OMIM: 209900]), is an autosomal
recessive disorder that is characterized by hydrometrocolpos, postaxial
polydactyly, and congenital heart disease [15–17]. The MKKS gene
encodes a 570-amino acid polypeptide with a group II chaperonin-like
domain; MKKS mRNA is widely expressed in many tissue types [18,19].
Wild-type (WT) MKKS protein rapidly shuttles between the centrosome
and the cytoplasm [20], and it plays an important role in cytokinesis [21].
Some MKKS protein variants with missense mutations that have been
found in patients cause the mislocalization and rapid degradation of
the MKKS protein [20,21]. Furthermore, MKKS protein forms a complex
with other group II chaperonin-like proteins, BBS10 and BBS12, and six
CCT chaperonins [22]. This complex mediates the assembly of the
BBSome, which transports vesicles to the cilia.
In this paper, we report the extended involvement of the uORFs in
the 5′-UTR of the human MKKS gene. MKKS gene produces a short tran-
script that encodes only uORFs using alternative polyadenylation sites
at the 5′-UTR in addition to a canonical long transcript that encodes
both uORFs and MKKS. In contrast to the uORFs, the polyadenylation
at the 5′-UTR did not contribute to repress the translation of the down-
stream MKKS ORF in our reporter system. Moreover, we found that
two uORF-derived proteins, uMKKS1 and uMKKS2, are translated and
translocated to the mitochondrial membrane in cultured human
cells. Our database search revealed that the presence of uORF specific
transcript is atypical in human transcriptome. These results suggest a
unique mechanism for production of uORF peptides in MKKS gene.
2. Materials and methods
2.1. Plasmids
The human MKKS cDNA was amplified by PCR using a human heart
cDNA library as a template with primers 5′-TTGAATTCCTCGCGCGA
CGCGAAGGTTG-3′ (the restriction enzyme recognition site is underlined)
and 5′-CGCCTCGAGCTCTTAGTTTTTATCTTC-3′. The EcoRI/XhoI-digested
PCR fragment was subcloned into the mammalian expression vector,
pCMV-SPORT (Invitrogen, Tokyo, Japan), which generated pCMV-MKKS.
The uORFs of uMKKS1 and uMKKS2, with both a C-terminal FLAG-tag
and the restriction enzyme sites, were sequentially amplified from
pCMV-MKKS with the primary primer sets: 5′-TTCTGCAGACTATGAGCC
TTCGGAACTTGT-3′ (uMKKS1-PstI-f) and 5′-CTACTTGTCGTCATCGTCTTT
GTAGTCCTTCCCCTGGATTTGGCT-3´ for uMKKS1; and 5′-TTCTGCAGGAG
ATGAAAAATACCAGTTGG-3′ (uMKKS2-PstI-f) and 5′-CTACTTGTCGTCAT
CGTCTTTGTAGTCCTCTTCAATACTCTTTTG-3′ for uMKKS2. The secondary
primer sets were uMKKS1-PstI-f and 5′-TTCTCGAGCTACTTGTCGTCA
TCGTCTTTG-3′ (FLAG-XhoI-r), and uMKKS2-PstI-f and FLAG-XhoI-r.
The PstI/XhoI-digested uMKKS1-FLAG and uMKKS2-FLAG fragments
were subcloned into the pCMV-SPORT vector, which generated pCMV-
uMKKS1-FLAG and pCMV-uMKKS2-FLAG, respectively. For C-terminal
myc-tagged constructs, uMKKS1 and uMKKS2 ORFs were amplified
from pCMV-MKKS with the following primer sets: 5′-GCGGATCCACTA
TGAGCCTTCGGAACTTG-3′ and 5′-GTTTCTAGAGCCTTCCCCTGGATTTGGC
TC-3′ for uMKKS1; and 5′-GCGGATCCGAGATGAAAAATACCAGTTGG-3´
and 5′-GTTTCTAGAGCCTCTTCAATACTCTTTTG-3′ for uMKKS2. Then each
BamHI/XbaI fragment was subcloned into the corresponding sites in the
pEF4/myc-HisB vector (Invitrogen), which generated pEF-uMKKS1-myc
and pEF-uMKKS2-myc, respectively. For the luciferase assays, the luc2
cDNA fragment from the pGL4.23[luc2/minP] vector (Promega, Tokyo
Japan) was digested with HindIII/XbaI and inserted into the correspond-
ing sites of the pGL4.74[hRluc/TK] vector (Promega), which generated
pTK-luc2. The MKKS 5′-UTR fragment was amplified from pCMV-MKKS
with the primers 5′-AATAAGCTTCTCGCGCGACGCGAAGGTTG-3´ and
2729
5′-GCTGGCATCTTCCATGGTACTTCAGGTGGTAACTAG-3′ and inserted
into HindIII/NcoI sites of the pTK-luc2 vector, which generated
pTK-WT-luc2.
The pTK-mt0-luc2, mt1-luc2, mt2-luc2, mt012-luc2, PS-luc2, and
mt012PS-luc2 constructs were generated from the pTK-WT-luc2
vector by PCR based site-directed mutagenesis. Briefly, the AUG
initiation codons of the three uMKKSs were altered to UUG (leucine)
codons, separately or together, and the three AAUAAA polyadenylation
signals in the 5′-UTR were simultaneously altered to AACAAG. All
constructs were confirmed by sequencing.
The pDsRed2-Mito vector was purchased from Clontech (Tokyo,
Japan) for mitochondrial labeling. For immunoprecipitation, the MKKS
ORF with or without the 5´-UTR was amplified from pCMV-MKKS
with the following primer sets: 5′-TTGAATTCCTCGCGCGACGCGAA
GGTTG-3′ and 5′-CCCTCTAGACCGTTTTTATCTTCAATAAC-3′ for MKKS
with the 5´-UTR; and 5′-GCGAATTCAATGTCTCGTTTGGAAGCTAAG-3′
and 5´-CCCTCTAGACCGTTTTTATCTTCAATAAC-3′ for MKKS without the
5′-UTR. Each EcoRI/XbaI fragment was subcloned into the correspond-
ing sites of pEF4/myc-HisB, which generated pEF-UTR-MKKS-myc
and pEF-MKKS-myc, respectively. Mutated UTR (UTRmt) was ampli-
fied from pTK-mt012-luc2 with the following primer set: 5´-TTGAATTC
CTCGCGCGACGCGAAGGTTG-3′ and 5′-CAAAAGAGTATTGAAGAGAGAT
CTGGC-3′ and subcloned into EcoRI/EcoRV sites of pEF-UTR-MKKS-myc
vector to generate pEF-UTRmt-MKKS-myc.
2.2. Antibodies
To prepare the uMKKS1 and uMKKS2 polyclonal antibodies,
we immunized rabbits with synthetic uMKKS1 (SSPVFQIPKNDD) or
uMKKS2 peptides (QRSAKQSVKFQS). The peptides were subcutaneously
injected into two rabbits with Freund's complete adjuvant every
2 weeks. The antisera were then subjected to affinity purification using
a CNBr Sepharose column coupled to the same peptide used for
immunization.
The following primary antibodies were used for western blot and
fluorescent immunocytochemistry in this study: anti-cytochrome c
mAb (clones 6H2.B4 for immunofluorescence and 7H8.2C12 for western
blot, BD Biosciences, Tokyo, Japan); anti-porin mAb (clone 31HL,
Calbiochem, Tokyo, Japan); rabbit anti-PHB2 polyclonal antibody [30];
goat anti-BBS6 polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA); anti-c-myc mAb (clone 9E10, Clontech); anti-FLAG M2
mAb (Sigma, Tokyo, Japan); rabbit anti-FLAG polyclonal antibody (used
for co-immunostaining with cytochrome c, Sigma); and anti-GAPDH
mAb (clone 6C5, Chemicon, Temecula, CA, USA). The secondary anti-
bodies were as follows: horseradish peroxidase (HRP)-conjugated anti-
mouse IgG (GE Healthcare, Tokyo, Japan), HRP-conjugated anti-rabbit
IgG (GE Healthcare) and HRP-conjugated anti-goat IgG (Santa Cruz
Biotechnology) for western blot; Alexa488-conjugated anti-mouse
IgG (Molecular Probes, Eugene, OR, USA) for FLAG M2; and myc
Cy3-conjugated anti-mouse IgG (Chemicon) for cytochrome c.
2.3. Cell culture and DNA transfection
Human cervical carcinoma cells (HeLa), human hepatoma cells
(HepG2), human osteosarcoma cells (U2-OS), and human fibrosarcoma
cells (HT1080) were maintained in Dulbecco's modified Eagle's
medium (D-MEM, Wako, Osaka, Japan) supplemented with 10% fetal
bovine serum (FBS, Japan Bioserum Co., Ltd., Nagoya, Japan), 100 U/ml
penicillin, and 100 μg/ml streptomycin (Invitrogen). Human neuroblas-
toma cells (SH-SY5Y) were maintained in D-MEM supplemented with
15% FBS and antibiotics. The cells were cultured at 37 °C under 5% CO2.
For DNA transfections, cells were seeded onto 35-mm poly-L-
lysine-coated glass-bottom dishes (Matsunami Osaka, Japan, for immu-
nocytochemistry), 6-well plates (for western blots), or 24-well plates
(for luciferase assays), and the plasmid DNA was transiently transfected
into the cells using Lipofectamine 2000 (Invitrogen) according to the
2730 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738
manufacturer's instructions. After incubation for 20–24 h, the cells were
subjected to analysis.
2.4. Immunocytochemistry
The transfected cells were fixed for 20 min at room temperature
with 4% paraformaldehyde in phosphate buffered saline (PBS, 0.01 M
phosphate buffer, 0.0027 M KCl and 0.137 M NaCl (pH 7.4)) and were
treated for 10 min with 0.2% Triton X-100 in PBS, followed by two
washes with PBS. The cells were incubated for 1 h at room temperature
with the primary antibodies diluted into PBS containing 2% FBS and
0.05% Tween-20 (PBS-FT). The cells were washed three times with
PBS and were incubated with the fluorescence-conjugated secondary
antibodies in PBS-FT for 1 h at room temperature. Then, the cells were
washed three more times with PBS and analyzed with a μRadiance
laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA).
2.5. Preparation and fractionation of mitochondria
Preparation and fractionation of mitochondria from HeLa cells was
performed as described previously [30]. For western blotting, 15–25 μg
of protein was used in each fraction.
2.6. Western blot and immunoprecipitation
Cell extracts were prepared with lysis buffer containing 10 mM
Tris-HCl (pH 7.0), 150 mM NaCl, 1% Na-deoxycholate, 1% Triton
X-100, 0.1% SDS, phosphatase inhibitor cocktail I and II (Sigma), and
a protease inhibitor cocktail for use with mammalian cells and tissue
extracts (Sigma). The cell lysate was mixed with sample buffer and
boiled for 5 min at 95 °C. The samples were separated by 16% tricine
SDS-PAGE for uMKKS1 and uMKKS2 and 10% SDS-PAGE for all
others. The proteins were transferred onto nitrocellulose membranes
(Hybond ECL, GE Healthcare) or polyvinylidene difluoride mem-
branes (Millipore, Tokyo, Japan) using a semi-dry transfer system.
Membranes were blocked for 1 h at room temperature with 5%
nonfat milk in PBST (20 mM NaH2PO4, 80 mM Na2HPO4, 100 mM
NaCl and 0.1% Tween-20) or 2% bovine serum albumin fraction V
(Roche, Tokyo, Japan) in TBS (50 mM Tris-HCl (pH 7.4), 0.137 M NaCl
and 2.7 mM KCl), and incubated for 1 h at room temperature with the
primary antibody diluted in blocking solution. After washing, the mem-
branes were treated with an HRP-conjugated secondary antibody for
1 h at room temperature. Proteins were detected by chemilumines-
cence with ECL detection reagents (GE Healthcare). Antibodies were
used at the following dilutions: anti-uMKKS1 (1:50), anti-uMKKS2
(1:50), anti-MKKS (BBS-6, 1:500), anti-myc (1:1000), anti-FLAG
(1:5000), anti-cytochrome c (1:100), anti-porin (1:2000), anti-PHB2
(1:2000), anti-GAPDH (1:3000), anti-mouse IgG (1:5000), and anti-
rabbit IgG (1:2000).
The transfected HeLa cells were suspended in RIPA buffer (20 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1% Na-deoxycholate and 1% Triton
X-100). After sonication for 4 min on ice, the lysates were centrifuged
at 4 °C for 15 min at 10,000 g to remove insoluble materials. Five
micrograms of anti-myc antibody and G-Sepharose protein were
added to the lysates and rotated at 4 °C overnight. After washing five
times, the immunoprecipitated complexes were separated on 10%
SDS-PAGE, followed by western blot analysis using an anti-MKKS
antibody.
2.7. RNA isolation and northern blotting
Total RNA was prepared using TRIzol-reagent (Invitrogen)
according to the manufacturer's instructions, followed by DNase treat-
ment. The extracted RNAs were further purified using an mRNA purifi-
cation kit (GE Healthcare). An equal amount of mRNA (3 μg) was
separated by electrophoresis on a 1.2% morpholinepropanesulfonic
acid (MOPS)-formaldehyde-agarose gel. RNA was transferred on a
Hybond-N + membrane (GE Healthcare) by capillary diffusion and
fixed by UV irradiation. [α-32P] dCTP-labeled DNA probes for the
MKKS ORF cDNA fragment (+1120 to +1627) and for the 5′-UTR
cDNA fragment (-649 to -1 in NM170784) were prepared using
Ready-To-Go DNA Labeling Beads following the manufacture's protocol
(GE Healthcare). β-Actin probe fragments prepared from HeLa cells by a
set of primers, which was previously described [31], were used as
control. Human adult tissue membranes (Ambion, Tokyo, Japan) were
used for human tissue northern blots.
2.8. 3′ RACE
To determine the 3′ end of both the polyadenylated and
unpolyadenylated RNAs, RNA ligase-mediated RACE (RLM-RACE) was
performed as described previously [32]. Briefly, to add a linker at the
3′-end of the RNA, total RNAs (1 μg) isolated from HeLa and HT1080
cells were ligated with RNA oligonucleotide 5′-CUGUACUCGAGAU
CAACCUGC-3′, which is phosphorylated at the 5′-end and modified
with an inverted dT at the 3′-end using T4 RNA ligase (Takara, Shiga,
Japan). The tagged RNA was used for the synthesis of the cDNA with
SuperScript III (Invitrogen), utilizing the complementary linker primer
5′-AGCAGGTTGATCTCGAGTACAG-3′ (linker-r). The cDNA reaction
mixture was diluted two-fold in TE (pH 8.0) and used for PCR. PCR
amplification was performed using Taq polymerase (Takara) under
the following conditions: 1.5 min at 94 °C followed by 30 s at 94 °C,
1 min at 54 °C, and 30 s at 72 °C (30 cycles). The primers used were:
5′-CTGGTGCGCAGGTACACTGAT-3′ and linker-r for the 5′-UTR; and
5′-CACAAGACTCACAACGACCCAGAAAG-3′ and linker-r for the MKKS
ORF. PCR products were subjected to 1% agarose gel electrophoresis.
After staining with SYBR-safe (Molecular Probes), the PCR products
were excised and inserted into the TA pGEM-T-easy cloning vector
(Promega). The polyadenylation site was determined by sequencing.
2.9. Luciferase assay and quantitative RT-PCR
HeLa transfections were performed as described above. Briefly, the
transfection mixture contained a total of 3 μg of DNA per 24-well
plate, including 0.1 μg of the firefly luciferase reporter plasmid, 10 ng
of Renilla luciferase reporter plasmid (pRL-TK internal control), and
2.9 μg of the test expression vectors or empty control vectors.
Cells were harvested 24 h after transfection, and luciferase assays
were performed using the Dual-Luciferase Reporter Assay System
(Promega) following the manufacturer's protocol. Chemiluminescence
was measured using a 2104 EnVision multilabel plate reader
(Perkin-Elmer, Yokohama, Japan). To control for transfection efficiency,
firefly luciferase values were normalized to the values for Renilla
luciferase. All values are presented as the mean ±SD calculated from
the results of three independent experiments.
For quantitative RT-PCR, total RNA was prepared from the cells 24 h
after transfection using TRIzol. One microgram of total RNA was used for
the synthesis of the cDNA with SuperScript III (Invitrogen) and a ran-
dom hexamer. The cDNA reaction mixture was diluted five-fold in TE
(pH 8.0) and used for PCR. The Renilla luciferase was used as an internal
control. Real-time quantitative PCR was performed in a LightCycler 1.5
system (Roche) using LightCycler FastStart DNA MasterPLUS SYBR
Green I (Roche). The primers used were as follows: 5′-GCTCAGC
AAGGAGGTAGGTG-3′ and 5′-ACCTTAGCCTCGAAGAAGGG-3′ for firefly
luciferase; and 5′-CTGATCTGATCGGAATGGGT-3′ and 5′-GACACTCTC
AGCATGGACGA-3′ for Renilla luciferase.
2.10. In vitro transcription/translation
The T7 promoter-based plasmids, pEF-UTR-MKKS-myc, pEF-UTRmt-
MKKS-myc and pEF-MKKS-myc, were used as the DNA templates. One
microgram of the DNA template was transcribed and translated in
 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738
50-μl reactions supplemented with 2.0 μl L-[ 35S] methionine
(1000 Ci/mmol at 10 mCi/ml, PerkinElmer) using quick-coupled
in vitro transcription/translation systems with the T7 promoter
(Promega), as described by the manufacturer's protocol. After
incubation at 30 °C for 2 h, 5.0 μl of the reaction mixture was
added into 20 μl of SDS sample buffer and heated at 80 °C for
5 min. Ten microliters of the denatured sample was used for the
16% tricine SDS-PAGE. The gel was dried and analyzed with a
Typhoon phosphor imager (GE Healthcare).
2.11. Statistical analysis
One-way analysis of variance (ANOVA) and Dunnett's post-hoc
test were performed using GraphPad Prism 5 (GraphPad Software,
Inc., San Diego, CA, USA).
2.12. Sequence and database analyses
Mitochondrial localization signals were predicted by MitoProt II
[33]. The prediction of the uORF and polyadenylation signal in the
5′-UTR was performed as following. We retrieved available human
cDNA sequences from RefSeq (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/
mRNA_Prot/human.rna.gbff.gz on 15 August 2011). The human
5′-UTR database was derived from the cDNA sequences containing
RefSeq corresponding coding sequences. Polyadenylation signals with
AATAAA or ATTAAA and uORFs were identified using two Java
programs, the “Polyar” [23], and the “Getorf” of the EMBOSS package
(version 6.3.1, The European Molecular Biology Open Software Suite),
respectively. For the search, we defined a uORF as a sequence with a
start codon within a 5′-UTR, an in-frame stop codon preceding the
end of the main coding sequence, and with a length of at least 60 nt
including the stop codon. The results were manually cured to obtain
the 5′-UTR databases including PS results with or without uORFs
presented in the Supplementary Data (in Excel file format). The search
for polyadenylation sites at the 5′-UTR was performed using the corre-
sponding UniGene EST database.
3. Results
3.1. Conservation of amino acids in the uORFs of the MKKS 5′-UTR
According to our analysis of the proteome of the mitochondrial
membrane proteins in HeLa cells, we detected a short protein that
was encoded by the 5′-UTR of the human MKKS gene (unpublished
data). The human MKKS gene consists of six exons with two alternative
5′-terminal exons that are regulated by alternative promoters (Fig. 1A).
Both mRNAs (1A: NM_170784 and 1B: NM_018848) contain several
uORFs within the 5′-UTR (Fig. 1B). We refer to the three longest
uORFs identified as uMKKS0, uMKKS1 (detected in the proteomic
analysis), and uMKKS2. uMKKS0, uMKKS1, and uMKKS2 encode pro-
teins that are composed of 43, 63, and 50 amino acids, respectively.
The predicted protein sequences of uMKKS1 and uMKKS2, but not
uMKKS0, are highly conserved in mammals (Fig. 1C). We did not
identify any sequence conservation of uMKKS1 or uMKKS2 in chickens
or fish (data not shown), despite the fact that the MKKS protein is
conserved among vertebrates [21]. The initiation codon context for
both uMKKS1 and uMKKS2 partially matches the optimal start codon
context at the -3 position [a strong start codon is indicated by a purine
at -3 and a G at the +4 position [3]. This suggests that uMKKS1 and
uMKKS2 may be translated into proteins in mammals.
3.2. The MKKS gene produces two types of mRNAs via alternative
polyadenylation
To verify whether the uORFs were present in the MKKS mRNA, we
performed northern blot analysis using a probe for the MKKS coding
2731
region (Probe 1) and the 5′-UTR region (Probe 2) (Fig. 2A). Using
the probe for the MKKS coding region one band that was approxi-
mately 3.2-kb long was detected (Fig. 2B, left) in poly(A) RNAs
purified from HeLa, U2-OS and HepG2 cells, which is consistent
with previous studies [18,19]. However, when we used the probe
for the 5′-UTR region, we observed two bands in each lane (Fig. 2B,
right). The slowly migrating band was the same size as the band
observed using Probe 1, which indicates a canonical mRNA that con-
tains the MKKS coding region and the 5′-UTR. The faster migrating
band running at approximately 0.8–1.1 kilobase (kb) in length that
was detected using Probe 2 has not been previously reported. We
detected both the long and short mRNAs in various human adult tis-
sues, except for brain tissue, by Northern blot using the 5′-UTR probe
(Fig. 2C). Therefore, the short mRNA is not restricted to cultured
human cells and is expressed almost ubiquitously in human tissues.
As the MKKS ORF is 1713 nucleotides (nt) in length, the short
mRNA is thought to be an mRNA lacking the full-length MKKS ORF.
The alternative promoters of the MKKS gene produce two types of
mRNAs with different 5′-UTR lengths of 759 nt (NM_170784) and
887 nt (NM_018848), which are approximately the same size as the
fast migrating band. There are three putative polyadenylation signals
in the 3′-terminus of the 5′-UTR and one in the first half of the MKKS
ORF (Fig. 2A). Therefore, we hypothesized that the short mRNA is
produced by the polyadenylation of the 5´-UTR. To determine the 3′
end of the MKKS mRNA, we performed 3′-rapid amplification of
cDNA ends (RACE) using total RNAs isolated from HeLa and HT1080
cells. Using a primer specific for the MKKS ORF, we detected a single
band that was 0.6 kb long in both the HeLa and HT1080 cells
(Fig. 2D, lanes 3 and 4). Sequence analysis of this band exhibited
the same polyadenylation site as was observed in NM_170784 and
NM_018848 (data not shown). In the analysis using a primer specific
for the 5′-UTR, we detected two bands that were 0.7 kb in HT1080
cells and 0.6 kb in HeLa cells (Fig. 2D, lanes 1 and 2). Sequence
analysis of these bands revealed polyadenylation at different sites
(Fig. 2E). The 0.7 kb band was polyadenylated after the second
polyadenylation signal (PS2) in the 5´-UTR (see Fig. 2A), and the
0.6-kb band initiated polyadenylation after the first polyadenylation
signal (PS1). To confirm the utilization of the polyadenylation signal
present in the 5′-UTR in vivo, we performed a search of human
expression sequence tags (ESTs) and a cDNA database library. We
found two types of utilization of the polyadenylation signals PS1
and PS3 (Supplemental data, List 1). These results indicate that the
MKKS gene produces both short and long polyadenylated mRNAs.
Alternatively, the MKKS protein is only translated from the long
mRNA that contains the uORFs.
3.3. uORFs, but not polyadenylation of the MKKS 5′-UTR, cause translational
repression of the downstream ORF
The presence of uORFs generally represses the translation of
the downstream ORF [4,10]. Further, polyadenylation of the MKKS
5′-UTR may block the expression of the main ORF. To understand
the role that the uORFs and the polyadenylation signals in the
5′-UTR play in the translation of the downstream MKKS reading
frame, we prepared a luciferase reporter constructs with a WT and
mutated MKKS 5′-UTR. The MKKS 5′-UTR was subcloned between
the thymidine kinase promoter and a firefly luciferase reading
frame. We constructed six types of mutant plasmids, which contained
mutations that disrupted either the uORFs or the polyadenylation
signal in the 5′-UTR. To eliminate the translational potential of the
uORF, an AUG to UUG mutation was introduced at the initiation
codon of uMKKS0, uMKKS1, and uMKKS2, or in all of three simulta-
neously, thereby generating the constructs mt0, mt1, mt2, and
mt012, respectively (Fig. 3A). To eliminate the polyadenylation signal
in the 5′-UTR, we altered three potential polyadenylation signals to
AACAAG, which generated mtPS. Further, we constructed a mutant
2732 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738

Fig. 1. The two uORFs, uMKKS1 and uMKKS2, present in the noncoding portion of the MKKS transcript are conserved among mammals. (A) The human MKKS gene consists of six
exons, in which exon 1 is selected by the alternative promoters, 1A (NM_170784) and 1B (NM_018848). The white boxes indicate the UTRs of an exon, shaded boxes indicate the
MKKS coding region, and lines represent the introns. Exon and intron lengths are displayed under the box and above the line, respectively. (B) The 5′-UTR of the MKKS transcript
contains six uORFs (black box) that are conserved in both transcripts by alternative promoters, and a specific uORF that exists in NM_018848 (gray box). There are three uORFs
encoding polypeptides that are >40 amino acids: uMKKS0, uMKKS1, and uMKKS2. The left numbers depict the three frames of MKKS cDNA, and +0 represents the main MKKS
reading frame. The uMKKS1 ORF is located in-frame to the MKKS ORF, whereas both uMKKS0 and uMKKS2 begin at the +2 position. Large shade boxes show the MKKS reading
frame. The uORFs are characterized by an AUG codon in the 5′-UTR, an in-frame stop codon, and a length of ≥9 nt, including the stop codon. (C) A comparison of the uMKKS1
and uMKKS2 amino acid sequences between humans (Homo sapiens, NM_018848), monkeys (Macaca mulatta, XM_001116188), mice (Mus musculus, NM_021527), and rats
(Rattus norvegicus, NM_001008353). Sequence alignments were performed using ClustalW 2.0.10 software.

(mt012PS) that contains mutations at each initiation codon of the
uMKKS and polyadenylation signals in the 5′-UTR. Next, constructs
containing the WT 5′-UTR of the MKKS gene or its derivatives were tran-
siently transfected into HeLa cells with the Renilla luciferase expression
plasmid. Twenty-four hours after the transfection, the amount of lucif-
erase RNA and activity were analyzed. The addition of the WT 5′-UTR
sequence greatly reduced both the mRNA levels and luciferase activity
compared with the UTR-deficient luciferase constructs (data not
shown). Each mutation in the uMKKS AUG codons (mt0, mt1, or mt2)
caused approximately a two-fold increase in the luciferase activity
when compared with the WT (Fig. 3C), although we did not observe
an increase in luciferase mRNA levels from these constructs (Fig. 3B).
Furthermore, the combination of the mutations enhanced the activity
by approximately six-fold (mt012), thereby suggesting that there
were additive effects exerted from each uORF. These results indicate
that the MKKS uORFs can repress the translation of the downstream
ORF, as previously described for other uORFs [4,10]. Additionally, each
MKKS uORF equally contributed the inhibition of translation, although
the sequence similarity between uMKKS0, uMKKS1 and uMKKS2 was
very low (data not shown). When the polyadenylation signals were
mutated, the luciferase mRNA levels increased by approximately
2.5-fold (Fig. 3B, mtPS), thereby indicating that the polyadenylation
signals in the 5′-UTR may affect the production of the long form of
the transcript. The luciferase activity was slightly reduced compared
with WT (Fig. 3C, mtPS). These data suggest that elevated mRNA con-
centrations due to the inhibition of polyadenylation make no contribu-
tion to the translation of the downstream ORF. When mt012PS was
transfected, we observed an increase in both mRNA levels and luciferase
activity. The increase in the luciferase activity was caused by the AUG
mutations of the uORFs in the 5′UTR. Taken together, these results
suggest that the hypotranslational rate of the downstream MKKS
reading frame is due to the presence of the uORFs but not the
polyadenylation of the 5′-UTR.
3.4. uORFs repress the translation of MKKS in vivo and in vitro
To confirm the influence that the uORFs exert on the translation of
MKKS, we constructed C-terminal myc-tagged MKKS expression plas-
mids either with or without the three uORF mutations in the 5′-UTR
(Fig. 4A). After transient transfection of either construct into HeLa
cells, myc-tagged MKKS protein (MKKS-myc) was detected by immu-
noprecipitation and western blot. MKKS-myc protein expression was
not detected in the transfections of either the empty or WT plasmids
(Fig. 4B). However, MKKS-myc protein (from a construct that did not
contain the MKKS 5′-UTR) was detected in HeLa cells, which suggests
that the presence of the 5′-UTR markedly suppresses the expression
of myc-MKKS protein. When the three AUGs of the uORFs in the
MKKS 5′-UTR were mutated, we detected MKKS-myc expression.
Thus, these in vivo results are consistent with the luciferase reporter
assay results mentioned above.
Furthermore, to examine whether the uORFs are translated as
peptides, we performed an in vitro coupled transcription/translation
assay using rabbit reticulocyte lysates with the same constructs
utilized in Fig. 4A. Using tricine-SDS-PAGE, we detected robust
expression levels of a single protein with an approximate molecular
weight of 62 kDa in the reaction mixture containing the UTR(−) con-
struct (Fig. 4C). Because the calculated molecular weight of MKKS-myc
is approximately 65.6 kDa, the 62-kDa band likely corresponds to the
MKKS-myc protein. When using the WT construct, we detected two
additional fast-migrating bands (7.1 kDa and 6.6 kDa, respectively) in
addition to the MKKS-myc protein (Fig. 4C, WT). We did not observe
these bands in the reaction mixture using the constructs without the
5′-UTR (UTR(−)) or with the disrupted uORFs (mt012). The calculated
molecular weights of uMKKS1 and uMKKS2 are approximately
7.26 kDa, and 5.80 kDa, respectively. Therefore, these peptides appear
to be synthesized from the uORFs of uMKKS1 and uMKKS2, but we
cannot eliminate the possibility of synthesis of uMKKS0 (calculated
 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738 2733

Fig. 2. The MKKS gene produces a short transcript that is polyadenylated in the 5′-UTR. (A) A schematic of the MKKS mRNA structure. The thin line indicates the probe region used
for northern blotting. The arrow indicates the primer-annealing sites used in the 3′-RACE assay. PS demonstrates the site of the polyadenylation signal sequence (AAUAAA) in the
MKKS mRNA (NM_170784). Four putative sites (−215 to −210, −64 to −59, −38 to −33, and 358 to 363 nt from the adenosine of the initiation codon of the human MKKS ORF)
and the canonical site were named PS1, PS2, PS3, PS4 and PS5, respectively. (B) Northern blot of mRNA from HeLa, U2-OS, and HepG2 cells using the MKKS ORF probe (probe 1, left),
the MKKS 5′-UTR probe (probe 2, right) and a β-actin probe (lower panels). The size of the RNA marker is indicated on the left. (C) Northern blot of mRNA from various human
tissues using probe 2 (upper panel) and a β-actin probe (lower panel). (D) RLM-RACE for the MKKS gene using the total RNA from both HT1080 (lanes 1 and 3) and HeLa cells
(lanes 2 and 4). To amplify the 3′-terminal fragment of the MKKS mRNA by PCR, primers for the 5′-UTR (lanes 1 and 2), the MKKS ORF (lanes 3 and 4), and the 3′-linker were
used as described in the Experimental procedures. (E) Sequence analysis of the fragments amplified by RLM-RACE using the 5′-UTR primer. Sequence of the 0.6-kb fragment
obtained from HeLa cells indicates that the polyadenylation site is downstream of PS1 (see Fig. 2A), whereas it is 0.7-kb downstream of PS2 in HT1080 cells. The box indicates
the polyadenylation signal, and the arrow indicates the polyadenylation site.

molecular weight is 5.02 kDa). Moreover, the AUG mutations in the
uORFs increased the synthesis of MKKS-myc protein compared with
the construct containing the WT 5′-UTR, which is consistent with our
in vivo results (mt012, compared with WT).
3.5. Transiently overexpressed uMKKS1 and uMKKS2 localize to
the mitochondria
The results of in vitro translation using rabbit reticulocyte lysates in-
dicated that the uORFs in the 5′-UTR of the MKKS gene are translated
into peptides in vivo. As the cellular functions of the uORF-encoding
products are poorly characterized, we examined the subcellular localiza-
tion of the uORFs in the 5′-UTR via indirect immunocytochemical
analysis. We focused on the uMKKS1 and uMKKS2 proteins, both of
which are highly conserved among mammalian species. Plasmids ex-
pressing C-terminal myc-tagged uMKKS1 (uMKKS1-myc) or uMKKS2-
myc were transiently transfected into HeLa cells. Immunoblot analysis
of the lysates derived from the cells expressing uMKKS1-myc or
uMKKS2-myc demonstrated efficient expression of both proteins
(Fig. 5B). Indirect immunostaining using an anti-myc antibody revealed
that both uMKKS1-myc and uMKKS2-myc co-localize with
co-expressing DsRed-Mito protein, a mitochondrial marker protein
(Fig. 5A). Furthermore, mitochondrial localization was verified by
co-localization of C-terminal FLAG-tagged uMKKS1 (uMKKS1-FLAG)
and uMKKS2-FLAG with endogenous cytochrome c in HeLa cells
(Fig. 5A,B, data not shown for uMKKS2-FLAG). These results indicate
that the transiently overexpressed uMKKS1 and uMKKS2 proteins
localize to the mitochondria.
3.6. Endogenous uMKKS1 and uMKKS2 are tightly bound to the
mitochondrial membrane
To determine whether the endogenous uMKKS1 and uMKKS2
proteins were translated in human culture cells, we prepared rabbit
polyclonal antibodies rose against the peptides from uMKKS1 and
uMKKS2. Western blot analysis using the anti-uMKKS1 antibody
revealed a single band with an apparent molecular mass of 7 kDa
from HeLa, U2-OS, and HepG2 cell extracts (Fig. 6A). The apparent
molecular mass corresponds to the predicted molecular weight of
uMKKS1 (~ 7.26 kDa). In SH-SY5Y cell extracts, the expression ratio
2734 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738

Fig. 3. The 5′-UTR of the MKKS gene inhibits the activity of the downstream luciferase via the uORFs but not the polyadenylation signal. (A) Constructs of the firefly luciferase
reporter with the WT MKKS 5′-UTR or its derivatives for the dual luciferase assay. Three uORFs, uMKKS0-2 and the polyadenylation sites (PS) in the MKKS 5′-UTR are illustrated.
For uORF disruption, the AUG codons of the three uMKKSs were changed to UUG either independently or simultaneously. To disrupt the polyadenylation signal, the AAUAAA
sequences of the three sites were changed to AACAAG. Bold X letters indicate a disrupted position of the uORF and/or the PS. (B) Quantitative RT-PCR of the luciferase transcript
in HeLa cells transfected with the reporter plasmids. Error bars represent the mean values ± SD of the relative luc mRNA levels from at least five independent experiments. The
asterisk indicates a significant difference from WT at P b 0.001 by one-way analysis of variance (ANOVA) followed by Dunnett's test. (C) Relative luciferase activity in HeLa cells
transfected with the reporter plasmids. Error bars represent the mean values ± SD of the relative luciferase signals from eight independent experiments in duplicate. All mutants
indicate significant differences from the WT (P b 0.01, ANOVA, Dunnett's post-hoc test).

of uMKKS1 to MKKS protein expression was low compared with the
other cell lines. In contrast to uMKKS1, we did not detect endogenous
uMKKS2 using the anti-uMKKS2 antibody in the whole-cell extracts
(data not shown).
We next determined whether uMKKS1 was present in the mito-
chondrial fraction. As expected, uMKKS1 was enriched in the same mi-
tochondrial fraction as cytochrome c (Cyt-c), a mitochondrial protein
(Fig. 6B). To determine the submitochondrial distribution of the
uMKKS1 protein, the mitochondrial fraction was divided into a mem-
brane pellet and a soluble supernatant by sonication. The submito-
chondrial fractions are controlled by prohibitin 2 (PHB2), a mitochon-
drial inner membrane-integrated protein; Cyt-c, a mitochondrial
inner membrane-associated protein; and porin, a mitochondrial
outer membrane-integrated protein. From subfractionation of the
mitochondria, endogenous uMKKS1 protein was detected in the mem-
brane pellet (Fig. 6C). Moreover, uMKKS1 was detected with PHB2 and
porin, but not Cyt-c, in a pellet following Na2CO3 treatment. This indi-
cates that endogenous uMKKS1 is a protein that is tightly bound to the
mitochondrial membrane. Interestingly, a 6-kDa band, which may possi-
bly represent endogenous uMKKS2 (~5.80 kDa), was identified in the
post-Na2CO3 pellet with the anti-uMKKS2 antibody (Fig. 6C). Detection
of the endogenous protein was likely due to protein enrichment by frac-
tionation and/or the removal of other impurities of a similar size. These
observations indicate that two of the uORFs in the MKKS 5′-UTR,
uMKKS1 and uMKKS2, are translated and integrated into the mitochon-
drial membrane in cultured human cells.
Next, we examined the amino acid sequences of the uMKKS1 and
uMKKS2 proteins to analyze their mitochondrial targeting signals

Fig. 4. Simultaneous uORF mutations upregulate the translation of MKKS in vivo and in vitro. (A) C-terminal myc-tagged MKKS expression constructs with the WT 5′-UTR (WT),
the uORF-mutated 5′-UTR (mt012, see Fig. 3A), or without the 5′-UTR (UTR(−)). (B) Immunoprecipitation of myc-tagged MKKS isolated from HeLa cells transfected with each
expression plasmid as described in (A). After immunoprecipitation using the anti-myc antibody, western blot analysis was performed using an anti-MKKS antibody. In the
UTR(−) lane, one-third of the volume of the precipitated sample was loaded. (C) MKKS-myc and uMKKS proteins were synthesized by coupled transcription/translation using
rabbit reticulocyte lysates in vitro. Proteins were separated by 16% tricine SDS-PAGE. Control lane (−) represents in vitro translation products in the absence of template. Positions
of the protein marker are shown on the left. Asterisks indicate synthesized uMKKSs.
 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738 2735

Fig. 5. Transiently expressed uMKKS1 and uMKKS2 localize to the mitochondria. (A) HeLa cells were transiently transfected with C-terminal myc-tagged uMKKS1 (upper panel), uMKKS2
(middle panel) or FLAG-tagged uMKKS1 (lower panel) with and without the mitochondrial marker protein DsRed-Mito (red). After fixing the cells, myc- and FLAG-tagged proteins were
immunostained with either anti-myc or anti-FLAG polyclonal antibodies, followed by incubation with Alexa488 labeled anti-rabbit IgG antibody (green). Fluorescence images were cap-
tured using a laser confocal microscope. In the lower panel, cells were immunostained with a cytochrome c antibody, followed by Cy3-labeled anti-mouse IgG antibody (red). Scale bar,
10 μm. (B) Western blot analysis of the transiently overexpressed uMKKS1 and uMKKS2 proteins. Whole-cell lysates were prepared from HeLa cells transiently overexpressing both
myc-tagged or FLAG-tagged uMKKS1 and uMKKS2. Samples were probed with myc (upper panel) or FLAG (third panel) antibodies. The GAPDH antibody was used as an internal standard.

(MTSs) using MitoProt II software (Table S1). The analysis revealed
that both uMKKS1 and uMKKS2 displayed high probability scores
for mitochondrial import (0.38 and 0.63, respectively). In contrast,
the amino acid sequences of uMKKS0 and MKKS displayed very
low probability scores for mitochondrial import (0.05 and 0.08,
respectively). These observations support the results of the mito-
chondrial localization experiments of both uMKKS1 and uMKKS2.
3.7. Polyadenylation signals at the 5′-UTR are mostly silent in human
uORF-containing genes
As shown in Fig. 2B and C, we identified the efficient expression of
an unexpected uORF-specific transcript, which is polyadenylated
using Polyadenylation signal (PS) located at the 5′-UTR in the MKKS
gene. We aimed to analyze whether PS localization to the 5′-UTR
region downstream of the uORF exists in other human transcripts.
We analyzed the human 5′-UTR dataset in the RefSeq database
using two programs, “polyar” for poly(A) signal prediction [23] and
“getorf” for uORF prediction (60 nt ORF including a stop codon [24].
From the 31,043 5′-UTRs including alternative transcripts, polyar pre-
dicted PSs in 749 sequences (Supplemental data, List 2), whereas the
getorf program identified uORFs in 9587 sequences. It was predicted
that 567 sequences contained both uORFs and PSs (1.8% in total,
Fig. 7A and Table S2). Of these 5′-UTRs, several sequences contained
multiple PSs or uORFs (87 sequences and 3009 sequences, respectively).
To search transcripts such as MKKS (i.e., that at least one uORF is located
in the region upstream of PS at the 5′-UTR) we compared the location of
the third nucleotide of the stop codon at the 5′-end uORF with the
location of the first nucleotide of the 3′-end PS in the 5′-UTR. Of the
transcripts containing both a uORF and PS, 333 sequences (1.1% in
total; Supplemental data, List 2(+)) were identified as transcripts in
which the 5′-end uORF was located in the region upstream of the
3′-end PS (uORF → PS), while 234 sequences (0.8% in total) were iden-
tified as transcripts in which the 3′-end PS was located in the region
2736 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738
Fig. 6. uMKKS1 and uMKKS2 are enriched in the mitochondrial membrane fraction.
(A) Western blot analysis of uMKKS1 and MKKS protein expression. Whole-cell extracts
were prepared from HeLa, U2-OS, HepG2, and SH-SY5Y cells. The samples (15 μg) were
probed with anti-uMKKS1 (upper panel), anti-MKKS (middle panel) and anti-GAPDH
(lower panel) antibodies. (B) Twenty-five μg each of whole cell, cytoplasmic extracts
(Cyto), the post-mitochondrial fraction (PMF) and the mitochondrial fraction (Mito)
were probed with anti-uMKKS1 (upper panel) and cytochrome c (Cyt-c, lower panel)
antibodies. (C) The mitochondrial fractions prepared from HeLa cells were sonicated
and separated into supernatant (Sup) and membrane pellets (MP) by centrifugation.
The membrane pellets were further treated with Na2CO3 and then separated into
supernatant (Sup) and precipitant (Ppt) by centrifugation. Samples were analyzed by
western blot using uMKKS1, uMKKS2, PHB2, Cyt-c, and porin antibodies. Note that
uMKKS2 was only detected in the sonicated-membrane and alkaline-treated pellets.
upstream of last nucleotide on the 5′-end uORF (both uORF and PS
except uORF → PS). In the group of uORF → PS, we found two MKKS
transcripts that came from NM_170784 and NM_018848. This indicates
that the prediction is effective.
As several of transcripts characterized with a uORF followed by a PS
were found in our bioinformatic prediction, we aimed to determine
whether the PS was functional in vivo. To evaluate this, we analyzed
human EST datasets available in UniGene. From this database search,
we selected 125 5′-UTRs (109 genes including MKKS gene; transcripts
with multiple PSs at the 5′-UTR were dominantly selected, Supplemental
data, List 3), which were all included in the uORF → PS group. Of the
tested sequences, approximately 80% indicated no evidence of tran-
scripts using a PS in the 5´-UTR in the human EST database. Interestingly,
we found that the MKKS gene displayed 33 EST hits that contained uORFs
and polyadenylation using the PSs in the 5′-UTR of the MKKS gene. These
EST database search results support our Northern blot and 3′ RACE ob-
servations, thereby indicating that the PS in the 5′-UTR of the MKKS
gene is effective in vivo. In the rest of sequences, we found that the num-
ber of identified EST was low compared with the EST number from the
MKKS gene (Fig. 7B). These suggest that PS located at the 5′-UTR is gen-
erally not used in vivo. Thus, the polyadenylation at the 5′-UTR in the
MKKS gene is likely a unique event in the human transcriptome.
4. Discussion
We demonstrated that the uORFs of the human MKKS gene repress
the translation of the main downstream MKKS reading frame. The
translational repression by the uORFs seems to be very robust because
when the level of mRNA increased by mutation of the polyadenylation
Fig. 7. Prediction of uORFs and/or polyadenylation signals in human 5′-UTRs. (A) The
ratio of the 5′-UTRs with uORF and/or PS. Transcripts with either or both uORF and
PS at the 5′-UTR were classified in percentage according to the result of PS or uORF
analysis using human 5′-UTR database with 31,043 entries in total (circle graph). We
further classified the 5′-UTRs involving both uORF and PS into two groups: transcripts
with the 5′ terminal uORF (5′-end uORF) followed by a 3′ terminal PS in the 5′-UTR
(3′-end PS) (5′-end uORF > 3′-end PS) and transcripts with a 3′-end PS followed by
the 5′-end uORF (3′-end PS ≥5′-end uORF). The former group includes the MKKS
transcript analyzed in our study. The full list of transcripts displaying a PS in the
5′-UTR is provided in the supplemental data. (B) The classification of number of the
EST/cDNA hits for transcripts indicating a 5′-end uORF > 3′-end PS.
signals at the 5′-UTR, the luciferase activity did not increase (Fig. 3).
The two longest uORFs, uMKKS1 and uMKKS2 (encoded by the long
and short MKKS transcripts), are highly conserved in mammals and
encode mitochondrial proteins that are expressed in various human
cell lines. It is unknown whether the third longest uORF (uMKKS0)
and the other smaller uORFs are translated in human cells. The uORFs
in the MKKS gene satisfy three of the four uORF properties for increased
translational inhibition [4]: i) evolutionary conservation, ii) increased
distance from the cap (average = 170 nt, > 750 nt in MKKS), iii) ex-
istence of multiple uORFs, and iv) a strong upstream AUG context
(a strong context is indicated by a purine at − 3 and a guanine at
+ 4 in relation to the upstream AUG). The AUG context of uMKKS1,
uMKKS2 and the main MKKS ORF only align at the -3 position. There-
fore, the AUG context of the uMKKSs in the long transcript likely af-
fects ribosome scanning for the AUG codon of the main MKKS
reading frame.
Many of the mutant MKKS proteins that both contain amino acid
transitions and are derived from a disease state are rapidly degraded
in cultured cells [20]. MKKS knockout mice display many clinical
findings that are also observed in human BBS patients [25]. This indi-
cates the importance of MKKS expression in the MKKS disease. Recent
studies on uORFs have proposed that the alteration of uORFs is asso-
ciated with some genetic diseases [4,9]. Our data suggest that the
existence of uMKKS0, uMKKS1, or uMKKS2 in the MKKS 5′-UTR
affects the expression of MKKS. Thus far, only one patient with a
mutation in the uORF of the 5′-UTR has been reported [26]. The
mutation was a G to A transition at the –74 position from the main
MKKS initiation codon, which resulted in the creation of a potential
initiation codon for a 4-amino acid uORF. However, the link between
MKKS pathogenesis and the uORF is still unclear.
 C. Akimoto et al. / Biochimica et Biophysica Acta 1830 (2013) 2728–2738
We demonstrated that the two longest uORFs in the human MKKS
gene are translated in vivo. In general, it is difficult to detect the en-
dogenously translated uORF products, because the average length of
a uORF in humans is generally short (48 nt, [4]). Therefore, the iden-
tification of an intact uORF product in mammalian transcripts has
been limited to mass spectrometric analysis [5]. A recent study on
the MYCN gene has revealed the existence of the endogenous
uORF-encoded protein, MYCNOT, using a rabbit polyclonal antibody
[27]. MKKS and uMKKSs, which are encoded on a polycistronic tran-
script, demonstrate distinct subcellular localizations. MKKS protein
shuttles between the centrosome and the cytosol [20], whereas
both uMKKS1 and uMKKS2 localize to the mitochondrial membrane.
The prediction of mitochondrial targeting using MitoProt II software
also supports the idea that uMKKS1 and uMKKS2 localize to the mito-
chondria. MKKS and uMKKSs likely exert different cellular functions.
An exogenously expressed uORF2 peptide in the glucocorticoid
receptor 1A transcript is localized to the inner side of the plasma
membrane, where it does not seem to interact with the glucocorticoid
receptor [28]. Thus, we predict that the peptides encoded by uORFs in
human transcripts function independently of their downstream
reading frame, but the number of small uORFs that are efficiently
translated is currently unknown.
uMKKS1 and uMKKS2 are potentially translated from a long
transcript containing MKKS reading frame and a short transcript pro-
duced by polyadenylation at the 5′-UTR. Although it remains unclear
which of the transcripts serves as a dominant template for the trans-
lation of the uMKKSs, the presence of a transcript for the uORF may
reflect the importance of the uORF peptide in regards to cellular func-
tion. According to our bioinformatic analyses, approximately 1% of
the human 5′-UTRs contains both a uORF and a downstream PS. How-
ever, the search results from the human EST database indicated a very
low frequency of use of the PS in the predicted 5′-UTRs. This implies
that the PS at the 5′-UTR is generally silent in human genes. In agree-
ment with this, a recent report has indicated that the PS becomes
silent in Drosophila and human cells when the PS localizes close to
the transcription start site [29]. Therefore, the polyadenylation at
the 5′-UTR of the MKKS gene seems to be a unique event, and the con-
sequent presence of a uORF-specific transcript may highlight the cel-
lular importance of the uMKKS proteins.
In conclusion, we demonstrated that the presence of multiple
uORFs inhibits the translation of MKKS, which is consistent with the
general function of uORFs as an RNA element. Furthermore, we have
shown that two uORFs in the MKKS gene transcript are translated
in vivo. The mitochondrial localization of the uMKKSs revealed that
proteins encoded by uORFs are functional in vivo. Therefore, our find-
ings provide extended insight into the function of mammalian uORFs.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.bbagen.2012.12.010.
Acknowledgements
We thank Ms. Chiyoko Kato and Ms. Eriko Ohta for superior experi-
mental assistance, members of the H.E. laboratory for valuable discus-
sions. This work was supported by a grant for Scientific Research from
the Ministry of Education, Culture, Science and Technology of Japan
(MEXT) [22590290 to H.E.], and from MEXT-Supported Program for the
Strategic Research Foundation at Private University. This publication
was also subsidized by JKA through its promotion funds from KEIRIN
RACE.
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