Download - Wiley Online Library

© 2016 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd
doi:10.1111/tra.12397
Human Fumarate Hydratase Is Dual Localized
by an Alternative Transcription Initiation
Mechanism
Ekaterina Dik1,† , Adi Naamati1,† , Hadar Asraf1 , Norbert Lehming2 and Ophry Pines1,2,∗
1 Department
of Microbiology Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel
Program and the Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore
2 CREATE-NUS-HUJ
∗
Corresponding author: Ophry Pines, [email protected]
Abstract
Fumarate hydratase (FH, fumarase), is a tricarboxylic acid cycle enzyme
sequence. On the basis of expression of endogenous wild-type FH and
localized in the mitochondrial matrix. However, a common theme, con-
mutant FH cDNAs from plasmids, RT-PCR, RACE to determine the 5′
served from yeast to human, is the existence of a large cytosolic pop-
termini of FH mRNAs, and mass spectrometry of FH products, we show
ulation of FH. FH has been shown to function as a tumor suppressor
that the mechanism of FH distribution is alternative transcription initia-
gene and is now implicated in various diseases. We have previously indi-
tion from a broad promoter. This is contrary to the suggested mechanism
cated that the cytosolic echoform of FH has a role in the DNA damage
for rat liver cells which had claimed alternative translation initiation.
response and specifically in the response to DNA double strand breaks.
In fact, recently FH has been shown to be involved in histone demethylation. Therefore, it has become important to understand the underlying
Keywords dual targeting, fumarate hydratase, mitochondria, nucleus,
transcription initiation, tumor suppressor
mechanism of FH dual subcellular location in human cells. We revealed
Received 12 November 2015, revised and accepted for publication 29
that in human cells, in contrast to yeast, the FH gene encodes two gene
March 2016, uncorrected manuscript published online 1 April 2016,
products, one containing and one lacking the mitochondrial targeting
published online 6 May 2016
Eukaryotic cells are defined by the existence of membrane
delimited subcellular compartments. Each compartment
contains a unique set of proteins, which execute specific
biological processes that are fundamental for the normal
performance of the compartment. Thus, protein targeting
to a specific location in the cell is an essential process for
the proper function and maintenance of the whole cell. In
eukaryotic cells, identical proteins can be localized to more
than a single subcellular compartment, a phenomenon
termed dual targeting. The two populations of the proteins are identical or almost identical and are termed
echoforms (1).
Dual targeting can be achieved by various mechanisms.
These mechanisms can be divided into two major subgroups, two and single translation product mechanisms.
Two translation products can be obtained by two mRNAs
from a single gene, either by alternative transcription initiation or by alternative splicing. In the case of a single
mRNA, the two translation products can be achieved by
alternative translation initiation or alternative translation
termination (2,3). In the cases of single translation products, about 10 different molecular mechanisms have been
described (3–7).
†
These authors contributed equally to this manuscript.
720 www.traffic.dk
A common theme conserved throughout evolution is the
dual localization of the enzyme fumarase, which in humans
is designated FH (fumarate hydratase). FH is distributed
Dual Targeting of Human FH
between mitochondria and the cytosol of all eukaryotes. In
mitochondria FH is essential for cell respiration as part of
the tricarboxylic acid (TCA) cycle. More than a decade ago
FH was found to underlie a tumor susceptibility syndrome,
hereditary leiomyomatosis and renal cell cancer (HLRCC).
This syndrome is characterized by benign cutaneous and
uterine leiomyomas, renal cell carcinomas and uterine
leiomyosarcomas. A bi-allelic inactivation of FH has been
detected in almost all HLRCC tumors, and therefore FH
was suggested to function as a tumor suppressor (8,9).We
discovered that extra-mitochondrial fumarase is required
for protection from double strand breaks, as part of the
DNA damage response (DDR), both in yeast and human
(10). We proposed that the role of fumarase as a tumor suppressor gene is because of its activity within the DDR. More
recently, Jiang et al. have shown that upon DNA damage,
FH is required to inhibit histone demethylases which is
crucial for efficient DNA damage repair (11). The histone
demethylases are one example for a superfamily of enzymes
called α-ketoglutarate-dependent dioxygenases. These
enzymes utilize α-ketoglutarate as a co-substrate, and are
involved in fatty acid metabolism, oxygen sensing, collagen
biosynthesis and modulation of the epigenome. Fumaric
acid is a competitive inhibitor of such enzymes and can
potentially modulate different cellular processes (12,13).
In addition, high concentrations of fumarate have been
shown to cause succination, which is an irreversible nonenzymatic modification of cysteine residues by fumarate, to
form S-(2-succinyl) cysteine (2SC). For instance, chronic
succination of glutathione has been associated with
persistent oxidative stress and cellular senescence (14).
Additional proteins that have been shown to be succinated
by fumaric acid are the Kelch-like ECH-associated protein
1 (KEAP1) and mitochondrial aconitase (ACO2) which
can have profound effects on cellular metabolism (15–17).
Finally, protein succination by fumaric acid has recently
been suggested to be involved in development of type 2
diabeties. In this regard, succinated mitochondrial proteins have been suggested as biomarker for type 2 diabetes
[(15,18,19) and Rorsman, unpublished data].
While the role of FH in health and disease has been the
topic of numerous studies, the distribution mechanism of
the enzyme in human cells remains undetermined. In rat
liver, the two echoforms are encoded by a single gene and it
has been claimed that it is transcribed into a single mRNA
Traffic 2016; 17: 720–732
which contains two translation initiation codons. The two
translation products presumably differ by the presence or
absence of the mitochondrial targeting sequence (20–23).
In the yeast Saccharomyces cerevisiae, fumarase is encoded
by the FUM1 gene and is expressed as a single translation
product which is distributed between the two compartments by a reverse translocation mechanism (24–29).
According to this mechanism all the protein molecules
start import into the mitochondria, during which the
mitochondrial targeting sequence (MTS) is removed by
the mitochondrial processing peptidase (MPP). After the
removal of the MTS, a subgroup of the molecules moves
back into the cytosol. The driving force of this process
is the folding of the protein (1,6,26–28). In Arabidopsis,
fumarase is encoded by two highly homologous genes
which differ by one containing and one lacking an MTS
(30).
In this study we first determined by a metabolic labeling
assay that, unlike yeast fumarase, human FH distributes by
a two translation product mechanism. Based on the analysis of FH distribution, when expressed from plasmids,
we conclude that FH does not distribute by an alternative
translation mechanism as suggested for rat liver. Finally,
by identifying the 5′ ends of FH mRNAs and by measurements of transcript levels we provide evidence that FH
distribution is determined at the level of transcription initiation, meaning that mitochondrial and cytosolic forms of
the protein are translated from different mRNAs. This conclusion is also supported by identification of FH N-termini
by mass spectrometry (MS) of wild-type and mutant FHs.
Results
FH has two translation products
In order to study the distribution mechanism of FH in
human cells, HeLa and HEK293T cell lines were employed.
First we examined whether FH is expressed as a single
translation product, as in yeast, or as two translation
products, as was suggested for rat liver. Analysis of the
FH sequence suggests the existence of an MTS at the N
terminus of the protein, which implies that this protein
is a substrate of MPP and is processed upon import into
mitochondria. We employed a metabolic labeling assay
in the absence and presence of the mitochondrial membrane potential uncoupler carbonyl CCCP. Uncoupling of
721
Dik et al.
can also be determined at the level of translation; two
products as above can be as a result of two translation
initiation events on a single mRNA transcript as suggested
for rat FH (20).
Figure 1: Human FH has two translation products.
Metabolic labeling with [35 S]-methionine of yeast and human
(HeLa) cells was carried out in the absence or presence of CCCP
(a mitochondrial membrane potential uncoupler which blocks
mitochondrial protein import). Fumarase/FH was immunoprecipitated from cell extracts using fumarase/FH antiserum which was
then analyzed using PAGE and autoradiography. P, precursor; M,
mature.
mitochondrial membrane potential blocks protein import
into the mitochondria and under these conditions we can
distinguish between the mitochondrial precursor which
contains the MTS from the mature processed form of the
protein. In the absence of CCCP only a ‘mature’ sized
form (lower arrow) of the protein can be detected, in both
yeast and human cells (Figure 1, lanes 1 and 3, bottom
panels). In the presence of CCCP we observe different
results for yeast and human cells. In yeast, we detect only
the precursor (upper arrow) that harbors the MTS which is
consistent with the production of a single translation product and the reverse mechanism of distribution (Figure 1,
lane 2, bottom panel). However, in human cells, in the
presence of CCCP, we detect two products, a precursor and
a mature sized protein (Figure 1, lane 4, bottom panel).
This clearly suggests, as anticipated from the study in rat
(20), a two translation product mechanism; one initially
long product, harboring the MTS, constitutes the mitochondrial population of the protein and a second shorter
product, lacking the localization signal which remains
in the cytosol.
The FH distribution mechanism is not at the level
of translation initiation
There are various mechanisms by which two translation
products can be dual distributed in the cell. Alternative
transcription initiation, will give rise to different transcripts, one with and one without the coding sequence for
the MTS. Alternative splicing or trans-splicing can remove
an MTS and also result in different mRNAs, one with and
one without the MTS coding sequence. The mechanism
722
Examining the FH gene sequence reveals that the first exon
encodes two in-frame ATGs, one at the beginning of the
exon and the second constitutes the last three nucleotides
of this exon (encoding M1 and M44, respectively, Figure 2).
Alternative splicing seems an unlikely mechanism for FH
distribution, because removal of the first exon would
eliminate the two possible translation initiation sites and
translation of the protein from any downstream AUG is
improbable, because it would eliminate at least 30 highly
conserved amino acids, which are crucial for the enzymatic
activity of FH (28).
In rat liver, the suggested mechanism for FH distribution is
alternative translation initiation from a single mRNA (20).
Consequently, based on these reports we first hypothesized
that alternative translation initiation, may be the mechanism of distribution of human FH. To approach this question we cloned the ORF of FH fused to a flag tag into a
pBabe-puro plasmid following the MO-MuLV 5′ LTR as the
promoter (32). The resulting plasmid pFH was transformed
into HEK293T cells. Expression and subcellular localization of the FH products were determined. In contrast to
the endogenous FH, when expressed from the pFH plasmid, harboring only the coding sequence of FH, the protein
is completely imported into the mitochondria as detected
by subcellular fractionation (Figure 3, compare the two
top left panels). Consistent with this conclusion, metabolic
labeling in the presence of CCCP shows only the precursor form of the protein is made and processed (Figure 3,
compare the two top right panels). Thus in contrast to the
endogenous gene, these results indicate that from these
plasmids all translation products of FH include the MTS.
The transcript of the plasmid-expressed-FH could lack
important elements of the endogenous FH mRNA. It is well
documented that the 5’ and 3′ UTRs affects the choice of
the translation initiation codon (33–35). In order to examine the influence of the addition of the 5′ UTR on the distribution of FH from a plasmid, we constructed a plasmid
containing an additional 60 nucleotides (nt) upstream to
the first AUG as was proposed by the genome browser
Traffic 2016; 17: 720–732
Dual Targeting of Human FH
Figure 2: Schematic illustration of the FH gene (not to scale). The FH gene has 10 exons represented by solid black rectangles.
Black lines between exons are introns and broken lines represent the upstream and downstream UTR regions. DNA: first and last
nucleotides of the first exon (+1 and +132), as well as the location of the last nucleotide (+267) of the second exon, are indicated by
arrows pointing up. Protein: The first and second in frame methionines (M1 and M44, respectively) are indicated with respect to their
amino acid location by arrows pointing down. The first five amino acids (starting with A45) encoded by the second exon are indicated,
the bold and underlined F is the first amino acid that is highly conserved from bacteria to human.
Figure 3: FH does not distribute by an alternative translation initiation mechanism. The schematic illustration of the FH
gene (not to scale), on the left, shows the FH ORF (FH), the FH mitochondrial targeting sequence (MTS), the 5′ and 3′ UTRs (60, 39
and 48 bp), the Met1 and M44 methionines and the flag tag. The 5′ and 3′ ends of the transcripts are according to genome browser
prediction (60 bp) or experimentally by RACE (39 and 48 bp, respectively). The panels on the right show the analysis of cells harboring
wild-type endogenous FH or FH expressed from the indicated plasmids. Cells were subjected to subcellular fractionation (left panels). The
anti-Hsp60 and anti-tubulin controls were preformed for all samples and the one shown is representative. [35 S]-methionine metabolic
labeling in the absence or presence of CCCP is shown in the right panels. P, precursor; M, mature; M1 and M2 are the two processed
forms of the M44V mutant. Antiserum: anti-FH; anti-flag; anti-tubulin (cytosolic marker); anti-Hsp60 (mitochondrial marker).
(http://genome.ucsc.edu/) for the FH 5′ UTR. The protein
expressed from this construct, again, did not distribute
and was completely imported into mitochondria (60 FH,
Figure 3, third panel).
Traffic 2016; 17: 720–732
Because there are only predicted UTRs described for
FH, we decided to determine these elements experimentally. For this we performed rapid amplification of cDNA
ends (RACE), followed by sequencing of the amplified
723
Dik et al.
products. We detected several 5′ UTRs of different lengths.
We assumed that the longest product contains all the
control elements required for distribution. The 5′ UTR of
FH that we detected contains 39 nt upstream of the first
ATG, whereas, the 3′ UTR was 48 nt long, of which the
terminal 16 nt constitute the poly A. We then re-cloned the
FH ORF according to the determined UTRs into the same
expression vector (Figure 3, p39 FH, fourth panel). Again
FH appears as a single translation product (fourth right
panel, right lane) and is not dual targeted residing exclusively in mitochondria (fourth left panel). Thus we were
unable to restore the distribution of FH by including the
UTRs. Worth mentioning is that some of these inserts were
also constructed in pTre2 puro or pCDNA3.1 expression
vectors under the CMV promoter (Figure S1, Supporting
Information). Thus our results were not expression vector
dependent.
To further test the hypothesis of alternative translation initiation as the FH distribution mechanism, we constructed
the above plasmids with the FH gene harboring specific
point mutations and examined the distribution of the corresponding plasmid-expressed-FHs. Mutagenesis of the
initiation codons (AUGs) resulting in M1V and M44V
(Figure 3). pM1V FH and pM44V FH eliminated the
first and second translation initiation codons, respectively.
FH M1V produced only the cytosolic echoform of FH, a
protein without the mitochondrial targeting sequence as
shown by subcellular fractionation and metabolic labeling
(Figure 3, pM1V FH, fifth right and left panels, respectively). This result is consistent with the scanning mechanism of translation initiation, according to which, the
ribosome scans the 5′ UTR until it reaches the first AUG
codon it encounters and starts translating from it (36–38).
The second mutation M44V resulted, as above with the
wild-type, in full import into mitochondria (Figure 3,
M44V FH, sixth panels). Unexpectedly we detected a new
protein product (M2 arrow) which has an apparent molecular weight between those of the mature (M1 arrow) and
precursor proteins (Figure 3, sixth right panel).This is
most probably due to alteration of the MPP recognition
site as will be discussed in more detail further on in this
manuscript. Finally we constructed a third mutant FH,
which contains an insertion of a single nucleotide between
the two AUGs, at position 69 of the nucleotide sequence.
This insertion results in a frame shift which should not
724
eliminate the ability of the ribosome to start translation
from this AUG1, but no expression of FH is expected from
this first AUG1 due to the downstream frame shift. This
construct allows us to test if the translation from the second AUG is possible in the presence of an intact first AUG.
As shown in Figure 3 (pFS FH, seventh panels) under
these conditions there is no expression of FH, implying that
translation initiation from the first AUG in a sense ‘rules
out’ translation initiation from the second. Taken together
we conclude that the FH distribution mechanism cannot be
explained by two translation products from a single mRNA.
According to Kozak (39) translation initiation can be
affected by the context of the AUG. The most important
position is −3 nt with respect to the A of the AUG and in
fact this is the nucleotide in the human FH. Consistent with
this argument, translation initiation from the first AUG of
the human FH mRNA is highly efficient without skipping
in vivo to the second AUG.
The FH gene has more than one type of transcripts
Once we concluded that a single mRNA mechanism is
unlikely to explain FH distribution, we asked whether there
is more than a single mRNA for FH in human cells. As
mentioned above, when we performed the 5′ RACE reaction we detected more than one sequence. Considering
this, we repeated the RACE reaction using a CapFishing
RACE kit which amplifies only 5′ capped mRNAs, thereby
increasing the specificity of the reaction. The reaction
products were cloned into an A/T cloning vector. Out
of the 90 plasmids extracted and sequenced, 22 were
‘empty’ (contained no sequence), whereas 68 provided FH
5′ sequences. Presented in Figure 4A are sequences that
repeated more than once. For each sequence the position of
the first nucleotide and the number of times each sequence
was found are displayed on x- and y-axes, respectively.
Displayed are 11 different 5′ ends of the FH mRNA that
encompass the region from 45 nt upstream of the first AUG
to 20 nt downstream of the second AUG. One exception is
an mRNA starting at position +205.
™
Not only the context of the sequence around the translation
initiation site is important but also the proximity to the cap
site. Recognition of the 5′ proximal AUG codon is inefficient when located too close the cap site causing utilization of AUGs further downstream (40). A leader sequence
Traffic 2016; 17: 720–732
Dual Targeting of Human FH
Figure 4: FH has more than one type transcript of transcripts. A) Graphic representation of all FH transcripts detected by the
™
CAP Fishing assay: After amplification of FH mRNA 5′ ends, cDNA products were cloned into an A/T vector and 68 sequences were
determined. The illustration bellow represents the location of each mRNA start point within the gene (x -axis) and number of times the
sequence was found (y-axis). B) Measurement of transcript levels of FH mRNAs: transcript levels were determined using ddPCR. Primer
sets were designed to amplify only transcripts that contain a specific region of the 5′ UTR (blue bar), first exon after the first ATG (after
ATG1, red bar) and second exon (after ATG2 green bar). Error bars indicate the Poisson 95% confidence intervals for each copy number
determination.
shorter than 15 nt is expected to be too short for proper
binding and activity of the ribosome [reviewed by (41)].
To simplify our analysis, we assumed that all transcripts
with 5′ UTRs longer than 18 nt are translated from the
first AUG, whereas for the shorter mRNAs the ribosome
does not initiate translation and continues to scan until it
reaches the second AUG. The transcript starting much further downstream (nt 205) is not expected to produce an
active enzyme and no protein products were detected to
support this short translation product (Figure 6), as will be
discussed later on.
The detection of two major groups of transcripts, which
according to our initial findings, can each generate only one
of the enzyme’s echoforms, supports a mechanism of distribution at the level of mRNA generation; transcription. It is
important to mention that none of the sequences displayed
any evidence for trans-splicing or splicing, such as non-FH
sequences or spliced FH sequences. This conclusion is supported using the MS analysis of FH products shown in the
following sections.
Additional proof that FH distributes by generation of different mRNAs, came from measuring the level of FH
Traffic 2016; 17: 720–732
transcripts using three sets of primers, each for a different
section of the FH coding sequence. The assumption is that
if there are mRNAs with different termini we should detect
variations in the levels of the different regions of the FH
transcript. For instance, expression from the first AUG is
only possible from mRNAs that contain the 5′ UTR region,
whereas the expression of the cytosolic echoform is only
possible when the first AUG is functionally absent.
We measured transcript levels by digital droplet PCR
(ddPCR) (42). This method involves a regular real-time
PCR reaction which is distributed across multiple reactions by packing the reaction mix and template within oil
droplets. This dilutes the DNA to a level at which some
reactions have no template and others that have one or
more template copies present. After amplification to the
terminal plateau phase of PCR, reactions containing the
template of interest yield positive endpoints, which are
detected by evagreen (Biorad) staining of the droplets. The
number of target DNA molecules present can be calculated from the fraction of positive endpoint reactions using
Poisson statistics. As ddPCR relies on a binary endpoint
threshold to assign each replicate reaction as either positive or negative (one or zero, respectively), it can tolerate
725
Dik et al.
wide variations in amplification efficiencies without affecting DNA copy number estimation. Because we used three
different primer sets, we could avoid the need to consider primer efficiency which by regular real-time PCR can
greatly influence the results (42).
Three sets of primers were applied which were designed
to recognize either he 5′ UTR, the first exon in proximity
to the second AUG, or the second exon. The assay was
repeated for two different cell lines, HeLa and HEK293T.
As shown in Figure 4B, in both cell lines tested, the level
of transcripts harboring the first AUG (5′ UTR primer set)
are significantly lower when compared with the level of
transcripts harboring the first exon (after AUG1). This
indicates that there is a subgroup of transcripts which
contain the second AUG but not the first one. These results
are consistent with the RACE results, demonstrating that
there are two types of mRNAs for FH, each of which
can give rise to one of the protein’s echoforms. Worth
mentioning is the fact that we see a third small group of
transcripts lacking both AUGs whose biological relevance
is unclear (‘205’, Figure 4A).
FH, expressed from its original promoter, distributes
by an alternative transcription initiation mechanism
The proposed distribution mechanism at the transcription level predicts that reconstitution of FH distribution
from a plasmid should occur by adding the genetic elements that control its transcription. We constructed a
plasmid containing 1000 bp upstream to the FH ORF.
As shown in Figure 5B (upper panel, lanes 1–3), the
presence of the promoter region resulted in FH distribution between the mitochondria and cytosol of the
‘p1000FH’ plasmid-encoded-FH. Metabolic labeling
detected two translation products, as previously shown for
the endogenous protein (Figure 5C, p1000FH, lanes 3 and
4), indicating distribution by a mechanism of alternative
transcription initiation. According to this mechanism,
each echoform is expected to be translated from a different
transcript. To test this model, we inserted a nucleotide
between the two AUG’s thereby creating a frame shift
mutation which should only disrupt the expression from
the transcript harboring the first AUG (of the mitochondrial echoform). In fact that is what we found, and unlike
the FS mutant, without the FH promoter, Figure 3; we
were able to detect a FH protein product from the 1000 FS
726
plasmid. As predicted this FH product is restricted to the
cytosolic fraction (Figure 5B, lanes 4–6) and metabolic
labeling detects a single product (Figure 5B, lanes 5 and 6).
Additional verification of these results is a M44V mutant
containing the promoter region, which as expected,
expresses only the mitochondrial protein (Figure S3).
Taken together, our results indicate that FH distribution is
determined at the level of transcription initiation.
Detection of the FH protein products
Up to this point in this study we examined the products
of the FH gene on denaturing polyacrylamide gels using
western blot or following labeling and immunoprecipitation. To obtain more precise information, we employed
MS with the intention of determining the N termini of
the different FH protein products. We isolated the endogenous enzyme by immunoprecipitation and determined its
N terminus after extracting the purified protein from an
acrylamide gel. A single N terminus was detected, ASQNSF
(Figure 6, endogenous FH).
The fact that we detected a single amino terminal peptide
starting immediately after the second methionine was surprising. The ASQNSF sequence result is consistent with
removal of the MTS by MPP between amino acid M44
and A45 as predicted by MitoProt (43). However, we
expected to detect a second N terminus, of the cytosolic
enzyme, starting with a methionine, MASQNSF, because
this cytosolic echoform is expressed from the second in
frame AUG. At this stage we assumed that a Met aminopeptidase (MetAP) could remove the first methionine. In this
regard MetAPs have been shown to require a short side
chain amino acid at the position immediately following the
N-terminal methionine (P1). Specifically for both MetAP1
and MetAP2 alanine is preferred (44,45) which in fact is the
amino acid following M44.
To gain support for the above notions we decided to look
at FH mutants that were referred to, in previous sections.
These mutant FHs were tagged with flag and overexpressed
from plasmids, purified and the samples were subjected to
N terminal labeling by dimethylation (31). This procedure
allows one to distinguish between the naturally occurring
N terminal in vivo, with those that may occur during the
preparation of the samples for the MS analysis. Translation
of M1V (the first AUG mutant), whose translation initiates
Traffic 2016; 17: 720–732
Dual Targeting of Human FH
Figure 5: FH distributes by an alternative transcription initiation mechanism. A) Schematic illustration of the indicated
plasmids. Bold black arrows represent the two translation initiation codons. MTS, FH, Met1, Met44 and flag are as indicated in the
legend to Figure 3.’Promoter’ indicates a 1000 bp FH sequence upstream of ATG1 (encoding Met1). The +C with the thin arrow
indicates insertion of a cytosine within the MTS encoding DNA sequence which creates a frame shift (FS) of translation initiating
from Met1 (ATG1). B) Subcellular fractionation of HEK293T cells harboring the indicated plasmids. The total (T), cytosolic (C), and
mitochondrial (M) fractions were analyzed using western blotting using the indicated antibodies. Controls: tubulin, cytosolic marker;
Hsp60, mitochondrial marker. C) Metabolic labeling ([35 S]-methionine) of HEK293T cells in the absence or presence of CCCP, with or
without the indicated plasmids. FH was immunoprecipitated with the indicated antibodies followed using PAGE and autoradiography
The MS analysis of plasmid-expressed-unmutated FH gave
both the ASQNSF sequence which is product of MPP processing and in addition at very low levels, a second N terminus ASAPGL (starting at amino acid A23, Figure 6, p39 FH,
second row). This was a rare peptide in the sample based on
both the frequency of the peptides detected starting at this
position (A23), and semiquantitation that was calculated
by the peak area of each peptide.
Figure 6: Determination of the FH protein N termini
and MTS. Endogenous genome encoded FH was purified from
HEK293T cell lysates by immunoprecipitation. The N terminus
was determined using mass spectrometry (MS) analysis. Plasmid
encoded FHs were purified from HEK293TFH−/− cells and were
subjected to N terminal labeling by dimethylation followed using
MS analysis. Antisera: anti FH, anti-flag.
only from the second AUG, resulted mainly the ASQNSF
N-terminus (Figure 6, M1V FH, third row). This result
supports our conclusion that the MASQNSF terminus is
processed by MetAPs. The MASQNSF peptide was also
detected for this mutant but at lower levels, which can
be explained by the plasmid-encoded-M1V-FH mutant
overexpression.
Traffic 2016; 17: 720–732
The second AUG mutant M44V produced the ASAPGL
N terminus as the more frequent peptide, along with significant amounts of the ASQNSF peptide. These results
indicate that by mutating M44 we affected the MPP processing. This is not surprising because the MPP cleavage
site is between M44 and A45, and replacing M44 with a
valine, changes the MPP recognition site. This change of the
MPP cleavage site allows a less preferred recognition site
(between L22 and A23) to be processed by MPP. The detection of high amounts of the longer form of FH (ASAPGL)
in this mutant, can also explain the novel high-molecular
band detected on SDS polyacrylamide gels for this mutant
(M2 band, Figure 3). In fact, we confirmed that the M1 and
M2 bands (Figure 3) of FH have the ASQNSF and ASAPGL
N-termini, respectively. Separate MS analysis was carried
out for each band.
727
Dik et al.
In all of the samples no shorter N termini were detected,
indicating that no AUG downstream to the second AUG,
is functional (Table S1). These MS results are in agreement
with our proposed mechanism of FH distribution. These
results also provide insight into events of processing, such
as MPP cleavage and Met-aminopeptidase cleavage.
Discussion
Here we explore the distribution mechanism of FH in
human cells. We show that the FH distribution mechanism
is at the level of transcription initiation, where for each
echoform separate mRNAs are made containing or lacking
a first functional AUG. The data supporting this conclusion are as follows (i) The FH ORF expressed from a plasmid with a defined promoter (Mo MuLV 5’LTR or CMV
promoter) initiating upstream of the first ATG, produces
only the mitochondrial protein. (ii) There is no expression
from the second AUG when the first AUG is present on the
mRNA (a frameshift mutation between the ATGs abolishes
all expression). (iii) A variety of transcripts were detected
both by ddPCR and RACE (both upstream and downstream of the first ATG). (iv) The FH ORF expressed from a
plasmid containing the promoter elements of FH (1000 bp
upstream of the first ATG) expressed the two echoforms
(p1000-FH). (v) The presence of the first AUG in this construct did not interfere with the expression of the second
shorter echoform whose transcription initiates from the
second ATG (p1000-FS).
In addition, the different N-termini that we detected by MS
support our model of an alternative transcription initiation
mechanism of dual targeting. According to our analysis
MPP cleaves between M44 and A45, whereas translation
of the shorter cytosolic echoform starts at M44. The MS
analysis provides insight as to the processing events which
FH undergoes. The molecular size of mitochondrial and
cytosolic FH echoforms, as can be detected using western blot and MS analysis, are identical. MS analysis of
the M1V mutant confirms the identity of the N-terminus
of the two echoforms which both start with A45. Without going into details here, the data indicate that the
N-terminal methionine of the shorter cytosolic echoform
is removed by methionine aminopeptidases. It is important
to note that no other N-terminal sequences were detected
which could support alternative splicing or translation
728
from downstream AUGs. Another point worth making is
that previous reports expressing FH from plasmids should
be revisited with regard to conclusions drawn, because we
expect that such expressed FHs are completely localized to
the mitochondria.
Our findings imply that the FH promoter region contains
the crucial information for FH dual targeting. The core
promoter is defined as the DNA segment of 50–100 bp
within which transcription initiates (46). This region is
required for recruitment of the transcription apparatus and
can be thought of as the priming stage for transcription
initiation. Promoters can be classified with respect to the
distribution of the transcription start sites (TSSs) they use.
‘Sharp’ class promoters use only one or a few consecutive
nucleotides as TSSs, resulting in a single-peak TSS distribution. These promoters often have TATA and/or initiator boxes and are correlated with tissue-specific genes
(47). A second group are ‘broad’ class promoters. For these,
transcription can initiate over a ∼150 bp region, resulting in a population of mRNAs that have different lengths
but usually the same protein-coding content. Broad promoters are often TATA-less and CpG-island-enriched and
are associated with housekeeping genes. Nevertheless, promoters containing both TATA boxes and CpG islands, as
well as promoters without any of these elements, have been
reported (48,49).
The FH promoter region contains a number of potential control elements. Of interest is a 639 nt sequence
that starts at position – 90 with regard to the first ATG,
and includes the first exon and part of the first intron.
This sequence has a high GC content and contains 76
CGs, thus it is considered a CpG island. CpG islands are
genomic stretches in which CG dinucleotides are overrepresented at significantly higher levels than is typical for
the genome as a whole. CpG’s in the promoter regions
are unmethylated CpGs, in contrast to methylated CpG
rich regions which are associated with gene silencing
(50). The existence of a CpG island in the promoter of
the FH gene, is consistent with our findings of multiple
TSSs for FH, suggesting a broad class promoter. A few
potential TATA-like regions were identified using a prediction analysis by Eukaryotic Promoter Database (EPD)
(http://epd.vital-it.ch/). However, these TATA boxes are
located more than 50 nt upstream to the longest 5′ UTR
Traffic 2016; 17: 720–732
Dual Targeting of Human FH
Figure 7: Schematic model of
human FH (fumarate hydratase)
distribution mechanism. A) FH is
transcribed from a single gene harboring
a broad type promoter. Transcription
starts from various transcription start
sites (TSS), creating different length
mRNAs. B) All mRNAs harboring a
sequence longer than 15 nt upstream
of the first AUG, will be translated from
the first AUG (left, orange arrow). These
will give rise to the MTS (mitochondrial
targeting sequence) harboring FH which
will be directed to the mitochondria. C)
mRNAs which are transcribed closer to
the first ATG or after, will be translated
from the second AUG (right, blue arrow).
that we identified while canonical TATA boxes should be
located 20–30 bp upstream of the TSS (51,52).
A schematic model of the FH dual distribution mechanisms summarized in Figure 7. The broad promoter (A)
leads to the transcription of different mRNAs which can be
divided into two groups, one with at least 15 nt upstream
to the first AUG indicated by orange arrows (B) that are
translated from the first AUG of the MTS. The second
group of mRNAs lacks the first AUG or lacks sufficient
length of 5′ UTR to be translated from the first AUG (less
than 15 nt). These mRNAs are translated from the second
AUG without the MTS and the protein products remain
in the cytosol (C).
The broad promoter of FH is expected for a house keeping gene which is required for respiration. The FH gene
represents unique use of a broad promoter, which allows
the generation of multiple mRNAs whose protein products are differently localized. This mechanism of FH dual
targeting in human, differs from suggested mechanisms
of this highly conserved enzyme in rat liver (translation
initiation), yeast (reverse translocation) and Arabidopsis
(two genes). Although the mechanism of dual targeting
may differ, the dual function of these enzymes appears to
be conserved (10). These findings are consistent with our
hypothesis that the evolutionary driving force for protein
Traffic 2016; 17: 720–732
dual targeting, is the function of each echoform in its
specific subcellular compartment, regardless of the mechanism by which the proteins are targeted to their destination
(53). Consistent with this line of thought is the extensive
diversity of mitochondrial targeting sequences (Figure S2)
while the fumarase structural protein sequences are highly
conserved.
Many transcription factors (TFs) have been reported to be
associated with the FH upstream sequence. According to
the USCS (University of California Santa Cruz) genome
browser (https://genome.ucsc.edu/) more than 70 different
TFs were detected in different ChIP assays for the 1000 nt
region upstream to FH. Future experiments will map this
region at a higher resolution in order to understand the
elements controlling the transcription of FH in human, and
whether we can distinguish between different transcription
initiation and control elements under different growth or
stress conditions (e.g. DNA damage).
In the recent years FH is the focus of an intensive research
along with other metabolic enzymes such as succinate
dehydrogenase (SDH) and isocitrate dehydrogenase
(IDH). It is becoming clear that the metabolites generated
by these enzymes can modulate the activity of various
enzymatic processes in the cell a are strongly related to
cancer prevention and development (54). Specifically, FH
729
Dik et al.
(fumarase) and fumarate have been implicated in various
cellular processes related to diseases such as oxidative
stress leading to mitochondrial dysfunction, succination leading to diabetes and DNA damage and genome
instability leading to cancer (10–12,15–19). Thus, our
contribution to the understanding of gene expression and
dual subcellular localization of this enzyme is important
for future studies.
Metabolic labeling of cell lines
Materials and Methods
tated with 15 μL anti-Flag affinity gel (Sigma A2220), anti-FH (AB110286)
and anti-Hsp60 antibody were diluted 1 of 100 and precipitated with 10 μL
Cell lines and growth conditions
protein A conjugated magnetic beads (Life technology 10002D). Beads
were washed with RIPA buffer, elution was done with 1× Laemmli sample
buffer. Samples were analyzed using SDS-PAGE and autoradiography.
HEK293T/17 [American Type Culture Collection (ATCC): CRL-11268],
HeLa (ATCC: CCL-2) cell lines were purchased from ATCC and grown
according to ATCC specifications in Dulbecco’s Modified Eagle Medium
(DMEM; D5671 sigma) with 10% FBS European grade (040071A Biological industries).
Plasmids
Human FH was amplified by polymerase chain reaction (PCR) from
human cDNA and a flag tag was added on the reverse primer. Untranslated
regions (UTRs) were also added on the primers. The 60 nt long 5′ UTR was
fused by a two-step PCR. The different fragments were inserted into three
different plasmids pBabe-puro, pTre2-puro and pCDNA3 as indicated.
All fragments inserted into pBabe-puro are between BamHI and SalI
restriction sites. Fragments were inserted into pTre2-puro between NheI
and ClaI restriction sites.
In order to clone the promoter site of FH preceding to the open reading
frame (ORF), the cytomegalovirus (CMV) promoter region of pCDNA3
Cells were grown in starvation medium DMEM/Hem’s nutrient mixture F12 (sigma 51445C) for 45 min × 10 μM 2Carbonyl CCCP was added
for 15 min. Starved cells were labeled with 40 μCi/mL [S35 ] methionine
and further incubated for 3 h. Cells were then harvested and lysed in
400 μL RIPA (Radioimmunoprecipitation assay) buffer (50 mM Tris–HCl
pH 7.4, 1% NP-40, 0.25% sodium deoxychlate, 150 mM NaCl, 1 mM EDTA
and protease inhibitor cocktail) for 30 min on ice. Cell debris was pelleted using centrifugation at 16 000× g for 15 min. The supernatant was
transferred to new tube and antibodies and/or beads were added for
overnight incubation at 4∘ C, as follows: Flag tagged proteins were precipi-
Cell fractionation
A total of 3 × 10 cm plates were grown to 70% of confluence, the media
was removed and cells were washed twice with ice cold PBS. Cells were
gently scraped and gathered into a single tube and spun down at 600× g for
10 min. The cell pellet was washed once in 1 mL of MIB (200 mM mannitol,
70 mM sucrose, 1 mM EGTA, 10 mM HEPES-KOH, pH 7.4, 1 mM DTT
and a protease inhibitor cocktail) and spun down at 600× g for 5 min.
The cell pellet was resuspended in 1 mL of MIB and incubated on ice for
5 min and then homogenized with 30 strokes of the Dounce homogenizer.
The homogenate was spun down at 600× g for 10 min in a pre-cooled
refrigerated centrifuge (4∘ C). The supernatants were spun down again and
a total (T) sample was taken from the supernatant before the next step. The
supernatant was spun at 10 000× g for 10 min at 4∘ C (the pellet contains
mitochondria while the supernatant contains cytosol and microsomes).
plasmid was removed by restriction with NruI and HindIII restriction enzymes, followed by a Klenow reaction and the purified plas-
A cytosol sample was taken from the supernatant and the pellet was
resuspended with 150 μL MIB. All samples where boiled in Laemmli
sample buffer.
mid was ligated. A two steps PCR amplified fragment including 1000 bp
upstream of FH was fused to FH encoding ORF these fragments were
cloned into the promoterless pCDNA3 using BamHI and XhoI restriction
Digital droplet PCR
sites.
Metabolic labeling of yeast cells
Wild-type yeast strain (BY4741) was culture to logarithmic phase in
galactose medium. The cells were harvested and labeled with 40 μCi/mL
[S35 ] methionine and further incubated for 30 min at 30∘ C. As indicated,
The ddPCR reaction was performed with 1 ng cDNA synthesized from
HeLa or HEK293T cells, 1 nM primer mix and QX200 ddPCR evagreen
supermix (Bio-Rad 186-4036). The reaction was performed in the Biorad
QX200 ddPCR system. Results were analyzed using the QUANTASOFT
software.
Primer sets that were used for amplification are: 5′ UTR: Forward:
20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added
before initiating labeling. Labeling was stopped by addition of 10 mM
sodium azide. Labeled cells were collected by centrifugation, resuspended
CAGCACCATGTACCGAG. Reverse: GCACCTTTAGTTCACCAAAG.
Exon 1: Forward: CGTGCCCTCGTTTTGG. Reverse: AATCCTGGTTTACTTCAGCG. Exon 2: Forward: CTTTGGTGAACTAAAGGTGC.
in Tris/ethylenediaminetetraacetic acid (EDTA) buffer pH 8.0, containing
1 mM phenylmethylsulfonyl fluoride, broken with glass beads for 5 min,
and centrifuged to obtain the supernatant fraction. Supernatants were
Reverse: CTCATCTGCTGCCTTCATTA.
denatured by boiling in 1% SDS, immunoprecipitated with anti-fum1 or
anti-Aco1 rabbit antiserum and protein A-Sepharose (Amersham Biosciences) and then analyzed using SDS-PAGE.
Race and Cap fishing assay
730
Smarter Race by Clontech and Seegene Cap fishing assay were performed
according to manufacturer protocol.
Traffic 2016; 17: 720–732
Dual Targeting of Human FH
Protein purification for MS
HEK293T cells were transfected with flag tagged plasmid. After 48 h
of transfection cells were harvested and lysed with Hepes-RIPA buffer
(50 mM Hepes pH 7.4, 1% NP-40, 0.25% sodium deoxychlate, 150 mM
NaCl, 1 mM EDTA and protease inhibitor cocktail). Purification preceded
according to Sigma protocol for flag tagged proteins using M2 flag affinity
gel. Elution was carried out with 100 μL 0.1 M glycine HCl, pH 3.5 which
was neutralized by adding 10 μL of 0.5 M Tris–HCl, pH 7.4, with 1.5 M
NaCl. Eluted proteins were subjected to N-terminus labeling by dimethylation (31) and then were analyzed using MS.
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Figure S1: Subcellular fractionation of FH-flag expressed from
pTre2-puro or pCDNA3 plasmids. Fractions were analyzed by PAGE
followed using western blot analysis. Anti-Hsp60, mitochondrial marker;
anti-tub (tubulin), cytosolic marker.
Figure S2: Multiple alignment of the N-terminal amino acid sequences
of fumarases from different organisms. Methionines (in bold) are colored as follows: blue, first methionine upstream of a mitochondrial targeting sequence (MTS); green, putative methionine for translation initiation
of fumarases lacking a MTS; red, amino acids that are highly conserved in
all organisms and which identify where the structural fumarase enzyme
sequence starts.
Figure S3: Subcellular fractionation of p1000M44V. Cells harboring the designated plasmids were subjected to subcellular fractionation.
Total (T), cytosol (C) and mitochondrial (M) fractions were analyzed
using western blot. Anti-Hsp60, mitochondrial marker; anti-tub (tubulin), cytosolic marker, anti-FH (fumarase); anti-flag.
Table S1: Mass spectrometry results of the N′ -terminal determination
of FH and the different mutants used in Figure 6.
References
1. Yogev O, Naamati A, Pines O. Fumarase: a paradigm of dual targeting
and dual localized functions. FEBS J 2011;278:4230–4242.
2. Schueren F, Lingner T, George R, Hofhuis J, Dickel C, Gartner J, Thoms
S. Peroxisomal lactate dehydrogenase is generated by translational
read through in mammals. eLife 2014;3:e03640.
3. Yogev O, Pines O. Dual targeting of mitochondrial proteins:
mechanism, regulation and function. Biochim Biophys Acta
2011;1808:1012–1020.
4. Avadhani NG. Targeting of the same proteins to multiple subcellular
destinations: mechanisms and physiological implications. FEBS J
2011;278:4217.
5. Kalderon B, Pines O. Protein folding as a driving force for dual protein
targeting in eukaryotes. Front Mol Biosci 2014;1:23.
6. Karniely S, Pines O. Single translation – dual destination: mechanisms
of dual protein targeting in eukaryotes. EMBO Rep 2005;6:420–425.
Traffic 2016; 17: 720–732
7. Yogev O, Pines O. Dual targeting of mitochondrial proteins:
mechanism, regulation and function. Biochim Biophys Acta 2011;
1808:1012–1020.
8. Launonen V, Vierimaa O, Kiuru M, Isola J, Roth S, Pukkala E, Sistonen
P, Herva R, Aaltonen LA. Inherited susceptibility to uterine leiomyomas
and renal cell cancer. Proc Natl Acad Sci USA 2001;98:3387–3392.
9. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D,
Leigh I, Gorman P, Lamlum H, Rahman S, Roylance RR, Olpin S, Bevan
S, Barker K, Hearle N, et al. Germline mutations in FH predispose to
dominantly inherited uterine fibroids, skin leiomyomata and papillary
renal cell cancer. Nat Genet 2002;30:406–410.
10. Yogev O, Yogev O, Singer E, Shaulian E, Goldberg M, Fox TD, Pines O.
Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear
component of the DNA damage response. PLoS Biol 2010;8:
e1000328.
11. Jiang Y, Qian X, Shen J, Wang Y, Li X, Liu R, Xia Y, Chen Q, Peng G,
Lin SY, Lu Z. Local generation of fumarate promotes DNA repair
through inhibition of histone H3 demethylation. Nat Cell Biol 2015;17:
1158–1168.
12. Nowicki S, Gottlieb E. Oncometabolites: tailoring our genes. FEBS J
2015;282:2796–2805.
13. Xiao M, Yang H, Xu W, Ma S, Lin H, Zhu H, Liu L, Liu Y, Yang C, Xu Y,
Zhao S, Ye D, Xiong Y, Guan KL. Inhibition of alpha-KG-dependent
histone and DNA demethylases by fumarate and succinate that are
accumulated in mutations of FH and SDH tumor suppressors. Genes
Dev 2012;26:1326–1338.
14. Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI,
Gaude E, King A, Leach JD, Edrada-Ebel R, Hedley A, Morrice NA,
Kalna G, Blyth K, Ruppin E, et al. Fumarate induces redox-dependent
senescence by modifying glutathione metabolism. Nat Commun 2015;
6:6001.
15. Merkley ED, Metz TO, Smith RD, Baynes JW, Frizzell N. The succinated
proteome. Mass Spectrom Rev 2014;33:98–109.
16. Ternette N, Yang M, Laroyia M, Kitagawa M, O’Flaherty L, Wolhulter
K, Igarashi K, Saito K, Kato K, Fischer R, Berquand A, Kessler BM,
Lappin T, Frizzell N, Soga T, et al. Inhibition of mitochondrial aconitase
by succination in fumarate hydratase deficiency. Cell Rep 2013;3:
689–700.
17. Adam J, Hatipoglu E, O’Flaherty L, Ternette N, Sahgal N, Lockstone H,
Baban D, Nye E, Stamp GW, Wolhuter K, Stevens M, Fischer R,
Carmeliet P, Maxwell PH, Pugh CW, et al. Renal cyst formation in
Fh1-deficient mice is independent of the Hif/Phd pathway: roles for
fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell
2011;20:524–537.
18. Blatnik M, Frizzell N, Thorpe SR, Baynes JW. Inactivation of
glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes:
formation of S-(2-succinyl)cysteine, a novel chemical modification of
protein and possible biomarker of mitochondrial stress. Diabetes
2008;57:41–49.
19. Frizzell N, Lima M, Baynes JW. Succination of proteins in diabetes.
Free Radic Res 2011;45:101–109.
20. Tuboi S, Suzuki T, Sato M, Yoshida T. Rat liver mitochondrial and
cytosolic fumarases with identical amino acid sequences are encoded
731
Dik et al.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
from a single mRNA with two alternative in-phase AUG initiation
sites. Adv Enzyme Regul 1990;30:289–304.
Akiba T, Hiraga K, Tuboi S. Intracellular distribution of fumarase in
various animals. J Biochem 1984;96:189–195.
Ono H, Yoshimura N, Sato M, Tuboi S. Translocation of proteins into
rat liver mitochondria. Existence of two different precursor
polypeptides of liver fumarase and import of the precursor into
mitochondria. J Biol Chem 1985;260:3402–3407.
Suzuki T, Sato M, Yoshida T, Tuboi S. Rat liver mitochondrial and
cytosolic fumarases with identical amino acid sequences are encoded
from a single gene. J Biol Chem 1989;264:2581–2586.
Ben-Menachem R, Tal M, Shadur T, Pines O. A third of the yeast
mitochondrial proteome is dual localized: a question of evolution.
Proteomics 2011;11:4468–4476.
Karniely S, Regev-Rudzki N, Pines O. The presequence of fumarase is
exposed to the cytosol during import into mitochondria. J Mol Biol
2006;358:396–405.
Knox C, Sass E, Neupert W, Pines O. Import into mitochondria, folding
and retrograde movement of fumarase in yeast. J Biol Chem 1998;
273:25587–25593.
Sass E, Blachinsky E, Karniely S, Pines O. Mitochondrial and cytosolic
isoforms of yeast fumarase are derivatives of a single translation
product and have identical amino termini. J Biol Chem 2001;276:
46111–46117.
Sass E, Karniely S, Pines O. Folding of fumarase during mitochondrial
import determines its dual targeting in yeast. J Biol Chem 2003;278:
45109–45116.
Stein I, Peleg Y, Even-Ram S, Pines O. The single translation product of
the FUM1 gene (fumarase) is processed in mitochondria before being
distributed between the cytosol and mitochondria in Saccharomyces
cerevisiae. Mol Cell Biol 1994;14:4770–4778.
Pracharoenwattana I, Zhou W, Keech O, Francisco PB,
Udomchalothorn T, Tschoep H, Stitt M, Gibon Y, Smith SM.
Arabidopsis has a cytosolic fumarase required for the massive
allocation of photosynthate into fumaric acid and for rapid plant
growth on high nitrogen. Plant J 2010;62:785–795.
Ji C, Guo N, Li L. Differential dimethyl labeling of N-termini of peptides
after guanidination for proteome analysis. J Proteome Res
2005;4:2099–2108.
Morgenstern JP, Land H. Advanced mammalian gene transfer: high
titre retroviral vectors with multiple drug selection markers and a
complementary helper-free packaging cell line. Nucleic Acids Res
1990;18:3587–3596.
Capell A, Fellerer K, Haass C. Progranulin transcripts with short and
long 5’ untranslated regions (UTRs) are differentially expressed via
posttranscriptional and translational repression. J Biol Chem
2014;289:25879–25889.
Jia J, Yao P, Arif A, Fox PL. Regulation and dysregulation of
3’UTR-mediated translational control. Curr Opin Genet Dev
2013;23:29–34.
Park J, Sharma N, Cutting GR. Melanocortin 3 receptor has a 5’ exon
that directs translation of apically localized protein from the second
in-frame ATG. Mol Endocrinol 2014;28:1547–1557.
732
36. Cigan AM, Feng L, Donahue TF. tRNAi(met) functions in directing the
scanning ribosome to the start site of translation. Science 1988;242:
93–97.
37. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic
translation initiation and principles of its regulation. Nat Rev Mol Cell
Biol 2010;11:113–127.
38. Kozak M. Regulation of translation via mRNA structure in prokaryotes
and eukaryotes. Gene 2005;361:13–37.
39. Kozak M. Pushing the limits of the scanning mechanism for initiation
of translation. Gene 2002;299:1–34.
40. Kozak M. A short leader sequence impairs the fidelity of initiation by
eukaryotic ribosomes. Gene Expr 1991;1:111–115.
41. Hinnebusch AG. Molecular mechanism of scanning and start codon
selection in eukaryotes. Microbiol Mol Biol Rev 2011;75:434–467,
first page of table of contents.
42. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ,
Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano
TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, et al.
High-throughput droplet digital PCR system for absolute quantitation
of DNA copy number. Anal Chem 2011;83:8604–8610.
43. Claros MG, Vincens P. Computational method to predict
mitochondrially imported proteins and their targeting sequences. Eur J
Biochem 1996;241:779–786.
44. Xiao Q, Zhang F, Nacev BA, Liu JO, Pei D. Protein N-terminal
processing: substrate specificity of Escherichia coli and human
methionine aminopeptidases. Biochemistry 2010;49:5588–5599.
45. Lowther WT, Matthews BW. Structure and function of the methionine
aminopeptidases. Biochim Biophys Acta 2000;1477:157–167.
46. Roeder RG. The role of general initiation factors in transcription by
RNA polymerase II. Trends Biochem Sci 1996;21:327–335.
47. Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, Stoeckert CJ
Jr. Promoter features related to tissue specificity as measured by
Shannon entropy. Genome Biol 2005;6:R33.
48. Roy AL, Singer DS. Core promoters in transcription: old problem, new
insights. Trends Biochem Sci 2015;40:165–171.
49. Sandelin A, Carninci P, Lenhard B, Ponjavic J, Hayashizaki Y, Hume
DA. Mammalian RNA polymerase II core promoters: insights from
genome-wide studies. Nat Rev Genet 2007;8:424–436.
50. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes.
J Mol Biol 1987;196:261–282.
51. Mathis DJ, Chambon P. The SV40 early region TATA box is required
for accurate in vitro initiation of transcription. Nature 1981;290:
310–315.
52. Ponjavic J, Lenhard B, Kai C, Kawai J, Carninci P, Hayashizaki Y,
Sandelin A. Transcriptional and structural impact of TATA-initiation site
spacing in mammalian core promoters. Genome Biol 2006;7:R78.
53. Kisslov I, Naamati A, Shakarchy N, Pines O. Dual-targeted proteins
tend to be more evolutionarily conserved. Mol Biol Evol 2014;31:
2770–2779.
54. Adam J, Yang M, Soga T, Pollard PJ. Rare insights into cancer biology.
Oncogene 2014;33:2547–2556.
Traffic 2016; 17: 720–732