We now show that internal translation initiation gives rise to E2F6b, an amino-terminal truncated E2F6 protein.. In this study, we now demonstrate that an amino-terminal truncated E2F6 p
Trang 1Two different E2F6 proteins generated by alternative splicing
and internal translation initiation
Tillman Dahme1, Jason Wood1, David M Livingston1and Stefan Gaubatz2
1
Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, USA;2Institute for Molecular Biology and Tumor Research (IMT), Philipps-University Marburg, Germany
E2F transcription factors play an important role in the
regulation of cell cycle progression E2F6, the most recently
identified member of the E2F family, is a
retinoblastoma-protein-independent transcriptional repressor that is
required for developmental patterning of the axial skeleton
It has recently been shown that the E2f6 locus produces two
different mRNAs, E2F6 and E2F6b The E2F6b mRNA
contains an additional exon that is inserted by alternative
splicing This exon contains an in-frame stop-codon and an
in-frame translation initiation codon However, whether a
protein is translated from the E2F6b mRNA has not yet
been addressed We now show that internal translation
initiation gives rise to E2F6b, an amino-terminal truncated
E2F6 protein We also show that E2F6 and E2F6b mRNAs are ubiquitously expressed in primary mouse tissues During the cell cycle, the highest expression of both forms is found at the G1 to S transition The 5¢ untranslated regions of E2F6 and E2F6b are unusually long, and they contain several upstream AUG codons followed by short reading frames Our results suggest that translation of E2F6b is initiated by internal ribosome entry We propose that regulated trans-lation initiation can produce distinct E2F6 isoforms under different physiological conditions
Keywords: E2F; transcription factor; cell cycle; alternative splicing; internal ribosomal entry
E2F transcription factors have been intensively studied for
their ability to control cellular proliferation E2F-responsive
elements have been identified in genes that play key roles in
cell cycle progression, synthesis of nucleotides, and DNA
replication (reviewed in [1]) Consequently, deregulation of
E2F can promote tumorigenic transformation (reviewed in
[2]) In normal cells, E2F activity is regulated by the binding
to pRB, the product of the retinoblastoma gene, and by
bind-ing to two related pocket proteins, p107 and p130 Recent
studies suggest that E2F proteins have a dual function in cell
cycle control First, free, uncomplexed E2F is a
transcrip-tional activator with growth promoting activities Secondly,
complexes between E2F and pocket proteins act as
tran-scriptional repressors and growth inhibitors [1]
E2F is a heterodimeric complex containing an E2F- and a
related DP- subunit So far, six E2F proteins (E2F1 through
E2F6) and two DP proteins (DP-1 and DP-2) have been
identified Conserved domains mediate DNA-binding,
dimerization, transactivation and pocket protein binding
In contrast to the other E2F proteins, E2F6, the most
recently identified E2F protein, lacks a transactivation
domain, and is a pocket protein independent transcriptional
repressor [3–6] We have recently shown that, in mice, E2F6
is required for developmental patterning of the axial skeleton
[7] E2f6 deficient animals display homeotic transformations
of the skeleton that are strikingly similar to the transforma-tions of certain polycomb deficient mice [7] These observa-tions are consistent with the recent finding that E2F6 associates with members of the mammalian polycomb complex [8,9] Taken together, these observations suggest that one function of E2F6 is to recruit polycomb multipro-tein complexes to target promoters during development
It has been recently shown, that the E2f6 locus produces two distinct mRNAs, E2F6 and E2F6b [10] The E2F6b mRNA contains the newly discovered exon 2 It has been noted that this exon introduces an in frame termination codon as well as an AUG codon on its 3¢ extremity, which could potentially serve as a translation initiation codon However, no evidence for the generation of a protein has been given
In this study, we now demonstrate that an amino-terminal truncated E2F6 protein is generated by internal translation initiation of E2F6b In addition, we show that the 5¢ untransl-ated region of the E2F6 mRNA is unusually long, and that they contain several upstream AUG codons followed by short reading frames, features that impair normal CAP-dependent translation initiation E2F6 and E2F6b mRNAs are widely expressed in primary mouse tissues We propose that regulated translation initiation can produce distinct E2F6 isoforms under different physiological conditions
M A T E R I A L S A N D M E T H O D S
Cell culture Cells were cultivated at 37C in a 10% CO2-containing atmosphere Cells were maintained in Dulbecco’s modified Eagle’s medium (Cellgrow) supplemented with 10% fetal bovine serum (HyClone) For the cell cycle experiment, MEF cells were serum starved in Dulbecco’s modified
Correspondence to S Gaubatz, Institute for Molecular Biology and
Tumor Research (IMT), Philipps-University Marburg,
35037 Marburg, Germany.
Fax: + 49 6421286 5196, Tel.: +49 6421286 6240,
E-mail: gaubatz@imt.uni-marburg.de
(Received 25 June 2002, revised 22 August 2002,
accepted 28 August 2002)
Trang 2Eagle’s medium/0.1% fetal bovine serum for 48 h, then
released from starvation by addition of Dulbecco’s modified
Eagle’s medium/10% fetal bovine serum The cells were
harvested at different time points after serum addition
Synchronization was confirmed by FACS analysis
RNA isolation
Total RNA was isolated from primary MEFs with the
RNeasy kit (Qiagen) according to the manufacturer’s
instructions To isolate RNA from primary mouse tissues,
an adult female C57/BL6 mouse was sacrificed, and then the
organs were harvested and quickly frozen in liquid nitrogen
Total RNA was isolated with TRIZOL (Invitrogen)
according to the manufacturer’s instructions
Luciferase fusion constructs
We first modified the sequence around the translation
initiation of the luciferase cDNA in pGL2-basic (Promega)
by introducing an AvrII-site and thereby abolishing the
initiating ATG, generating pGL2-Avr A fragment
enco-ding for the first eight codons of E2F6 and partial 5¢UTR
was amplified by PCR with primers SG108 and SG92 using
a genomic E2f6 clone as a template The resulting product
was digested with BglII and XbaI and inserted into
pGL2-Avr digested with BglII and pGL2-AvrII In a second step, a 2.4-kb
genomic SacI–BglII fragment containing the remaining
5¢UTR and 2.2 kb of the E2F6 promoter was inserted to
generate Exon1-luc A similar strategy was used to generate
Exon3-luc and Exon2-luc We first used RT-PCR of total
RNA with primers SG92 and SG122 to amplify part of the
coding regions and 5¢ UTRs of E2F6 and E2F6b mRNAs
PCR-products were digested with BglII and XbaI and
inserted into pGL2-Avr Secondly, the 2.4 kb genomic
SacI–BglII fragment was inserted to generate Exon2-luc
and Exon3-luc In Exon2-mut-luc, the translation initiation
codon was modified by PCR with primers SG92 and SG142
and Exon2-luc as a template The resulting PCR product
was digested with BglII and XbaI and inserted into
pGL2-Avr Finally, the 2.4 kb genomic promoter fragment was
inserted as described above An expression plasmid for
E2F6b was generated by PCR with primers SG109 and
SG35 The PCR product was digested with BamHI and
EcoRI and inserted into pcDNA3 All constructs were
confirmed by DNA sequencing Other plasmids have been
described previously: E1B-luc [11], E2F1-luc [12]
Polyclonal antibodies and immunoprecipitations
A polyclonal serum was raised in rabbits with KLH-coupled
peptide N-14 (CMSQQRTARRLPSLL) The antiserum
was affinity purified on columns with immobilized peptide
(Sulfolink, Pierce) and eluted in 100 mM glycine (pH 2.5)
The C-10 antiserum has been described previously [7] For
immunoprecipitation, MEFs were metabolically labeled
for 12–14 h with 1 mCi [35S]methionine and then lysed in
Tris/NaCl/NP-40 (50 mM Tris, pH 7.4, 150 mM NaCl,
0.5% NP-40, 1· complete protease inhibitors (Roche),
1 mM dithiothreitol) Lysates were incubated with 1 lg
affinity-purified antiserum Proteins were collected on
Protein-A Sepharose beads, washed five times in Tris/
NaCl/NP-40 and then separated by SDS/PAGE
Oligonucleotides SG24: 5¢-GGGAATTCTCAGATAGAGTCTTCTCTGG GAGC-3¢
SG50: 5¢-GGGGACCAGGGTGACCGCGG-3¢
SG92: 5¢-GCGCGGGAGATCTAACGGACGG-3¢ SG108: 5¢-CCTCTAGACGCGCCGTCCGGTGCTGAC TCAT-3¢
SG109: 5¢-GGGGATCCATGCCATCAAAAATAAGGA TTAAT-3¢
SG142: 5¢-CCTCTAGATTAATCCTTATTTTTGATGG CCCCCTTCTGTCTCTGCCTCCCAAGGACTGGC-3¢ SG35: 5¢-CGGAATTCCCCGTGCTGGAGGCGACT CG-3¢
SG122: 5¢-CGGACGGCGCGGAGAC 3¢
b-actin fw: 5¢-TGTGATGGTGGGAATGGGTCAG-3¢ b-actin bw: 5¢-TTTGATGTCACGCACGATTTCC-3¢ RT-PCR
PCR was performed with the Superscript One-Step RT-PCR kit (Invitrogen) with 100 ng of total RNA For the detection of E2F6 mRNA with primers SG122 and SG24, the following conditions were used: 1 cycle: 30 min, 50C; 1 cycle: 2 min, 94C; 35 cycles: 30 s, 94 C, 30 s, 55 C;
1 min, 72C, followed by 1 cycle for 10 min at 72 C For the detection of b-actin, the number of cycles was reduced to
32 Products were separated on 1.4% agarose gels Transient transfections and reporter assays Cells (2· 104) were plated per each well of a 24 well cell culture dish 24 h later, cells were transfected in triplicate using the indicated amount of luciferase fusion construct or empty vector and 3–5 lL of Fugene (Roche) diluted in
100 lL Dulbecco’s modified Eagle’s medium per triplicate reaction To analyze the transcriptional properties of E2F6b, U2-OS cells were transfected with 0.200 lg of E2F-dependet luciferase reporter construct (E2F1-luc or E1B-luc), 0.050 lg of CMV-bGal (to monitor transfection efficiency), and 0.200 lg mE2F6, pCDNA3-mE2F6b or pcDNA3-DP2 expression plasmids Cells were harvested 48 h after transfection and lysed in 1· passive lysis buffer (Promega) Luciferase assays were performed on
a Dynex Luminometer Luciferase activity was normalized
to b-Gal activity to correct for differences in transfection efficiency
Primer extension Total RNA was extracted from mouse embryonic fibro-blasts using the RNeasy mini kit (Qiagen) SG50 primer was end labeled with [c-32P]ATP and T4 polynucleotide kinase For the primer extension reaction, 10 lg of RNA was hybridized to radiolabelled SG50 at 65C for 90 min in hybridization buffer (0.15M KCl/0.01M Tris/Cl, pH 8.3/
1 mMEDTA) Hybridized RNA was then extended for 1 h
at 42C by addition of 30 lL reaction mix [1 lL 1MTris,
pH 8.3, 1 lL 0.5M MgCl2, 1 lL 0.25M dithiothreitol, 3.33 lL 2 mMdNTP mixture, 22.67 lL H2O, 1 lL 1 : 20 diluted Superscript II reverse transcriptase (Invitrogen)] RNA was subsequently degraded by incubating with
105 lL RNase (100 lgÆmL)1 sonicated salmon sperm
Trang 3DNA/20 lgÆmL)1 RNase A) for 15 min at 37C A
sequencing reaction was performed with the same SG50
32P-end-labeled primer using Sequenase 2.0 (United States
Biochemical) and the pBS-Not/Xho4.5 template The
sequencing products and the primer extension product were
separated on a QuickPoint gel (Novex) and visualized by
autoradiography
R E S U L T S
Two distinct mRNAs are produced from the E2f6 locus
It has recently been shown that the E2f6 locus produces two
alternatively spliced isoforms [10] (see Fig 1) The larger
splice variant is generated by insertion of an additional
exon 2 Exon 2 contains a stop codon, in-frame with the
upstream E2F6 sequence The last triplet of exon 2,
however, is a potential translation initiation codon (AUG), immediately upstream and in-frame with exon 3 Thus, it is possible that the E2F6b mRNA gives rise to an amino-terminal truncated protein, which is initiated from the internal AUG in exon 2 A second possibility is that a protein is synthesized by a read-through of the stop codon
in exon 2 The latter would give rise to a protein of slightly higher molecular mass containing 22 additional amino acids
The E2F6 5¢ untranslated region impairs ribosomal scanning
Translation of eukaryotic mRNAs is usually initiated at the first AUG triplet downstream of the 5¢-terminal CAP, and it
is usually separated from it by 50–100 nucleotides [13] Nevertheless, translation initiation from internal start sites
by internal ribosome entry is not uncommon Certain features, which are predicted to impair ribosome recruit-ment and linear scanning, have been found in the 5¢ untranslated regions (5¢UTRs) of mRNAs favouring inter-nal ribosome entry These features include a long 5¢UTR, stable secondary structures, and potential upstream initi-ation codons [14] To investigate whether E2F6 has a 5¢UTR that could promote protein synthesis from an internal AUG, we determined the site of transcription initiation by primer extension analysis (Fig 2) A fragment containing 3 kb of genomic sequences upstream of the E2F6 coding sequence was subcloned and partially sequenced Mouse E2F6 EST clones start approximately
Fig 1 Genomic organization of mouse E2f6 and the E2F6b splice
variant Blackboxes represent coding exons The open box represents
the 5¢ untranslated region of exon 1 (see Fig 3) The sequence of
exon 2 is shown below Translation termination and initiation codons
are indicated.
Fig 2 Mouse E2F6 promoter and 5¢ untranslated region (5¢UTR) (A) Identification of the E2F6 transcriptional start site by primer extension analysis Primer extension analysis was performed with radiolabelled primer SG50 (right lane) A genomic clone was sequenced with the same labelled primer, and the reaction was resolved on the same gel together with the primer extension reaction (left lanes) For primer location see (B) (B) Sequence of the mouse E2F6 promoter and 5¢UTR The 650 bp nucleotide sequence 5¢ of the transcriptional start site (+1) is shown The translation initiation codon is at +457 Upstream AUG codons are boxed The location of the SG50 primer used in the primer extension reaction is shown (C) Schematic representation of upstream initiation codons and of corresponding open reading frames in E2F6 and E2F6b Blacklines represent open reading frames with AUG codons in a context that favors translation initiation Grey lines represent other open reading frames Translation initiation of E2F6 is at +457 Translation of E2F6b is initiated at +628 Numbers represent location of initiation and termination codons relative to the transcription initiation site.
Trang 4200 base pairs upstream of the E2F6 open reading frame,
suggesting a minimum length of 200 nucleotides for the
5¢UTR of E2F6 (not shown) For primer extension analysis,
we chose a backward primer in the untranslated region that
anneals close to the beginning of the known EST clones (see
Fig 2B, primer SG50) Total RNA was isolated from
primary MEFs, then radiolabelled primer SG50 was
hybridized to the RNA and extended with reverse
tran-scriptase Sequencing reactions with the same radiolabelled
primer and the subcloned genomic template were resolved
on the same gel with the primer extension reaction We
observed a single band in the primer extension reaction
corresponding to a transcriptional start site about 457 base
pairs upstream of the translation start in Exon 1 (Fig 2A)
The presence of a single primer extension product indicates
that transcription of E2F6 and E2F6b is initiated at the
same position Therefore, the length of the E2F6 5¢UTR is
about 457 nucleotides, and that of the leader of the E2F6b
mRNA up to the potential translation initiation codon in
exon 2 is about 628 nt The sequence of the mouse E2F6
5¢UTR with the transcriptional and translational start sites,
as well as the partial promoter sequence, are shown in
Fig 2B We noted that there are three upstream AUG
triplets in the 5¢UTR of E2F6, and five upstream AUGs in
E2F6b (Fig 2C) None of the start codons have an optimal
Kozaksequence The third AUG at position +224,
however, is in a context, which is favourable for translation
initiation However, it would only allow the generation of a
short polypeptide terminated by in-frame stop codons
Furthermore, the 5¢UTRs of E2F6 and E2F6b mRNAs are
predicted to form extensive secondary structures by the
M-FOLDprediction software [15,16], as expressed in DGs of
)225.7 kcalÆmol)1 for E2F6¢s and )270.9 kcalÆmol)1 for
E2F6b’s 5¢UTRs Taken together, our findings strongly
oppose efficient translation initiation by ribosomal scanning
for both E2F6b and E2F6, and suggest that their translation
may be initiated internally
Two different E2F6 proteins accumulate in cells
If translation of E2F6b is indeed initiated at the internal
AUG in exon 2, it will give rise to an amino-terminal
truncated protein lacking the first 36 amino acids of E2F6
This protein would be predicted to be of a smaller molecular
mass than the previously described E2F6 protein To
identify a potential N-terminal truncated E2F6 protein, we
used two different polyclonal antisera directed against
oligopeptides derived from the C-terminus (C10) [7] and
from the amino-terminus (N14) of murine E2F6 (see
Fig 3A) C10 is predicted to recognize both forms of
E2F6, while N14 will only recognize the full-length E2F6
protein The specificity of C10 and N14 antisera was
confirmed by immunoprecipitation-Western experiments
with in vitro translated proteins (Fig 3B) Interestingly,
lysates of MEFs derived from E2F6 deficient mice lacked
two E2F6 specific bands, as compared with lysates of
wildtype MEFs in a Western blot probed with C10
antiserum (data not shown, but see [7]) In these
experi-ments, a second, slightly faster migrating protein was
detected in wildtype MEFs but not in E2F6 deficient MEFs
[7] Unfortunately, neither Western blots, nor
immuno-precipitation-Western blots of MEF lysates, probed with
N14 antiserum, revealed any E2F6 specific signal that could
be unambiguously distinguished from background (data not shown) We therefore employed an approach that turned out to be of higher sensitivity and specificity, and immunoprecipitated E2F6 from lysates of metabolically labelled MEFs In these experiments, a common band corresponding in size to E2F6 was detected by the C10 and N14 antisera (Fig 3C) Importantly, an additional, faster migrating protein was immunoprecipitated by C10, but not
by the N14 antiserum, which is specific for the full-length E2F6 variant The two proteins immunoprecipitated from cellular lysates by C10 antiserum correspond in size to
in vitro translated E2F6 and E2F6b proteins when com-pared to molecular mass standards (compare Fig 3B,C) Because of the lower affinity of the N14 antiserum, we cannot completely exclude the possibility that the band in the C10 immunoprecipitation corresponding in size to E2F6b is a proteolytic breakdown product To address this possibility it will be necessary to generate a higher affinity antiserum that is specific for the E2F6 protein
Translation of E2F6b is initiated at the internal AUG codon
To verify that the smaller E2F6b protein was the predicted E2F6b, we generated a reporter construct in which the luciferase coding sequence was fused in frame to the E2F6b coding sequence (Exon2-luc, see Fig 4A) In this construct, the E2F6b N-terminal coding sequence replaces the initi-ation codon of the luciferase Thus, luciferase enzyme activity can only be generated if translation is initiated within the E2F6b sequence The mRNA transcribed from
Fig 3 E2F6b gives rise to an amino-terminal truncated protein that is initiated from an internal initiation codon (A) Schematic representation
of the E2F6 and E2F6b protein structure The location of the peptides used to generate polyclonal antisera (C10 and N14) is schematically indicated (blacklines) (B) Characterization of the C10 and N14 antisera E2F6 and E2F6b were in vitro translated, and then subjected
to immunoprecipitation and Western-blotting with the indicated antisera Note that the C10 antiserum recognizes both E2F6 forms, whereas the N14 antiserum is specific for the full length E2F6 protein When E2F6b was in vitro translated, two E2F6b bands of slightly different mobility were observed (C) E2F6 was immunoprecipitated from lysates of metabolically labelled MEFs with the affinity purified E2F6 specific antisera C10 and N14, as indicated C10 precipitates two proteins that correspond in size to E2F6 and E2F6b, while N14 recognizes only one protein that corresponds to E2F6 Because of the lower affinity of the N14 antiserum, we cannot completely exclude the possibility that the band in the C10 immunoprecipitation that corres-ponds in size to E2F6b is a proteolytic breakdown product.
Trang 5this construct contains exon 1, exon 2, the first seven triplets
of exon 3, and thereafter the luciferase coding sequence
lacking only the initiating AUG Two constructs that lack
exon 2 served as controls In Exon3-luc, the luciferase
coding sequence was introduced after the seventh codon of
exon 3, which is the same fusion point as in Exon2-luc
Since there is the possibility that this rather long
amino-terminal E2F6 sequence fused to the luciferase will influence
its activity, a second control that only contained the first
seven codons of exon 1 (Exon1-luc) was used Importantly,
all luciferase constructs contain the complete E2F6 5¢UTR,
and their transcription is driven by a 2.2-kb fragment of the
E2F6 promoter (see Fig 4A) Transient transfection assays
in U2-OS cells revealed dose-dependent activity of
Exon2-luc which was two to three times lower than that
of Exon1-luc and Exon3-luc (Fig 4B) However, activity of
Exon2-luc was still up to more than 1000 times higher than
the activity of the empty vector, pGL2-Avr (Fig 4B, right
lanes) Similar results were found in NIH-3T3 cells (data not
shown) We therefore concluded that a protein is expressed
from the E2F6b mRNA
Our results suggest that translation of Exon2-luc is
initiated internally Alternatively, it is possible that a protein
is generated by a read-through of the stop codon in exon 2
To examine which mechanism is involved in the synthesis of
this protein, we mutated the internal AUG in exon 2 to
GGG (to generate Exon2-mut-luc), and compared its
activity to the activity of Exon2-luc in transient transfection
assays The mutation led to complete loss of activity in these
assays, indicating that the integrity of the AUG is crucial for
translation of E2F6b (Fig 4C) Taken together, this strongly suggests that translation can be initiated at the internal AUG in exon 2 of the E2F6b mRNA
E2F6 and E2F6b are ubiquitously expressed
It has previously been reported that E2F6 is ubiquitously expressed in mouse tissues [10] To analyze the relative expression of E2F6 and E2F6b in a larger panel of mouse tissues, we established a semiquantitative RT-PCR strategy Total RNA was isolated from multiple tissues of an adult C57/Black6 mouse and subjected to reverse transcription followed by PCR amplification with the primer set SG122 and SG24 E2F6 and E2F6b mRNA expression is shown in Fig 5A (top) Beta-actin specific primers were used as a control in a separate RT-PCR reaction (Fig 5A, bottom) Both E2F6 and E2F6b mRNA were detected in all tissues examined The highest expression levels of E2F6 and E2F6b were found in heart and skeletal muscle In most tissues, E2F6 levels were higher or equal to E2F6b However, one exception was skeletal muscle, where E2F6b was several fold more abundant than E2F6 (Fig 5A, right lane)
To analyze whether the expression of E2F6 and E2F6b
is cell cycle dependent, MEFs were serum starved for 48 h, and then released from starvation by the addition of serum Total RNA was isolated at three-hour intervals In addition, we isolated RNA from asynchronously growing cells, and from confluent, contact inhibited cells E2F6 and E2F6b expression was again analyzed by RT-PCR with
Fig 5 Expression of E2F6 and E2F6b (A) Expression of E2F6 and E2F6b in primary mouse tissues was analyzed by RT-PCR with primers SG122 and SG24 (top) RT-PCR with b-actin specific primers was used as a control (B) Expression of E2F6 and E2F6b during the cell cycle Mouse primary fibroblasts were brought to quiescence by incubation for 48 h in serum free medium, and then released into the cell cycle by the addition of 10% serum RNA was isolated at the indicated times after the addition of serum, and E2F6 and E2F6b expression was analyzed by RT-PCR with primers SG122 and SG24 RT-PCR with b-actin specific primers was used as a control Expres-sion in confluent (confl.) and asynchronously growing cells (asynchr.) was also analyzed The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle at each time point was determined by FACS analysis and is shown at the bottom.
Fig 4 Translation of E2F6b is initiated at an internal initiation codon.
(A) Schematic representation of the E2F6-luciferase fusion constructs.
The SacI site at )2.2 kb was used for cloning of the E2F6 promoter
(see Fig 1) Transcription initiation (+ 1) is indicated by a right arrow
(see Fig 2) (B) Activity of Exon2-luc (Ex2-luc), Exon1-luc (Ex1-luc),
and Exon3-luc (Ex3-luc), compared to the activity of the empty vector
(pGL2-Avr) Plasmids were transiently expressed in U2-OS cells, and
luciferase activity was determined Plasmid inputs (ng DNA) are
indicated Activity of pGL2-Avr at 100 ng was set to 1 (C) Activity of
Exon2-mut-luc compared to Exon2-luc after transient expression in
U2-OS cells Activity of Exon2-luc at 100 ng was set to 1.
Trang 6the primer set SG122 and SG24 (Fig 5B, top) Cell cycle
synchronization was monitored by FACScan analysis of
parallel samples (Fig 5B, bottom) E2F6 and E2F6b
mRNA levels were lowest in serum-starved cells During
re-entry into the cell cycle, E2F6 and E2F6b transcription
increased with peaklevels in late G1/early S Moderate
levels of the two transcripts were found in confluent and
asynchronously growing cells The relative abundance of
the two E2F6 mRNAs did not change significantly during
the cell cycle
E2F6b is a transcriptional repressor
Although E2F6b lacks the amino-terminus of E2F6, it is
otherwise identical in sequence to E2F6, except for the
initiating methionine derived from exon 2 (see Fig 3A)
Notably, E2F6b contains the DNA-binding and
dimeriza-tion domains, suggesting that E2F6b will bind to DNA and
dimerize with DP proteins like the previously described
E2F6 protein Previous workhas shown that E2F6 is a
pocket protein independent transcriptional repressor [3–6]
The repression domain of human E2F6 is localized in the
C-terminus of the protein [5] In contrast, in mouse E2F6
(also termed EMA), a repression domain has been identified
in the amino-terminus [3] Since E2F6b lacks the first 36
amino-terminal amino acids, we wanted to determine the
properties of E2F6b as a transcriptional regulator To
address this issue, we performed transient transfection
assays Two different reporter plasmids were utilized, in
which luciferase expression is under the control of the
p14ARFand the E2F1 promoters Both promoters contain
E2F sites and have previously been shown to be regulated in
an E2F-dependent manner [11,12] p14ARF and E2F1
luciferase reporter plasmids were cotransfected with E2F6
and E2F6b expression plasmids As expected, E2F6 reduced
activity of both promoters by about 50% to 60% (Fig
6A,B) Surprisingly, E2F6b also reduced the activity of these
promoters, although it was slightly less efficient than E2F6
To rule out the possibility that sequestration of the
dimerization partner DP is responsible for the reduction
in luciferase activity, we coexpressed DP2 together with
E2F6 and E2F6b (Fig 6A,B, + DP2) Coexpression of DP2 resulted in even further reduction of reporter activity, indicating that repression by E2F6b is not a result of titration of endogenous DP proteins Taken together, these results show that E2F6b is also a transcriptional repressor
D I S C U S S I O N
E2F6, the most recently identified E2F protein, is a retinoblastoma-protein independent transcriptional repres-sor In mice, E2F6 is required for developmental patterning
of the axial skeleton [7] Together with the recent finding that E2F6 associates with polycomb proteins [8,9], these observations suggest that E2F6 recruits polycomb com-plexes to certain target promoters during development It has recently been reported that the E2f6 locus generates two different mRNAs, E2F6 and E2F6b [10] However, no evidence for the generation of a protein from the E2F6b mRNA was given Interestingly, exon 2 in E2F6b contains
a stop codon, in-frame with the upstream E2F6 sequence mRNA species that contain premature termination codons are often destroyed by nonsense mediated decay so that only full-length proteins are produced [17] However, not all mRNAs that contain premature termination codons are targeted for destruction At the moment it is not clear how the E2F6b splice variant escapes nonsense mediated decay
In this study, we present evidence that a truncated E2F6 protein, E2F6b, is produced from the alternatively spliced mRNA by internal initiation of translation First, we show that an amino-terminal truncated E2F6 protein is present in cellular lysates of mouse embryonic fibroblasts Secondly,
we found the following structural features in the 5¢ leaders of E2F6b that make translation initiation by a ribosome-scanning mechanism unlikely: (a) a long 5¢ untranslated region, (b) stable secondary structure, and (c) potential upstream initiation codons The presence of these features in the E2F6b mRNA strongly suggests that translation of this protein is not initiated by a normal CAP-dependent mechanism Finally, with a set of luciferase-reporter constructs, we demonstrate that an internal initiation codon
is required for translation of E2F6b Translation initiation from internal start codons is usually not compatible with normal CAP-dependent ribosomal scanning In conclusion, these data strongly suggest that E2F6b is initiated by internal ribosome entry
We noted that in an earlier study, the transcription start site of E2F6 was mapped to 256 and 241 nucleotides upstream of the E2F6 AUG codon [10] In contrast, we found a single start at 457 nucleotides upstream of the AUG It is possible that the RNase-protection approach used by Kherrouche et al is more sensitive to extensive mRNA secondary structure than our primer extension strategy Alternatively, it is possible that the transcription start site of E2F6 is tissue dependent
In agreement with an earlier report, we found that the E2F6b mRNA is ubiquitously expressed in a wide variety of tissues The highest expression levels were found in heart and skeletal muscle In most tissues, except for skeletal muscle and heart, E2F6 was more abundant than E2F6b However, in another study, for some of those tissues reverse ratios between E2F6 and E2F6b were found [10] It is possible that this discrepancy is due to the different primers used for the reverse transcription reaction While we used a
Fig 6 E2F6b is a transcriptional repressor U2-OS cells were
trans-fected with 200 ng E2F-dependent reporter plasmids E2F1-luc (A)
[12], or with E1B-luc (with the p14 ARF promoter) [11] (B), and with
200 ng E2F6, E2F6b, or DP2 expression plasmids, as indicated 50 ng
CMV-b gal was cotransfected, and luciferase activity was normalized
to b-galactosidase activity Basal activity of the reporter plasmid in
presence of empty expression vector was set to 1.
Trang 7gene specific primer, an oligo(dT) primer was used in the
previous study We also show that the ratio between the two
E2F6 mRNAs does not change significantly during the cell
cycle
The E2F6b protein lacks the amino-terminal 36 amino
acids derived from exon 1 It shares with E2F6 the
DNA-binding and dimerization domain, as well as the C-terminus
Consequently, E2F6b is predicted to bind to DNA, and to
dimerize with DP proteins in a manner similar to E2F6
Surprisingly, E2F6b, like E2F6, is a repressor of E2F-site
dependent transcription (Fig 6), despite the fact that a
repression domain has been assigned to the amino terminus
of mouse E2F6 [3] It is possible that E2F6b specifically
represses some, but not other E2F-dependent promoters
in vivo Further experiments will be necessary to address this
possibility
Internal ribosome entry is known to still be functional
under conditions in which the usual CAP-dependent
mechanism is inactive [18–20] Examples include translation
of c-myc, which is mediated by an internal ribosome entry
site during apoptosis [18], and efficient translation of
vascular endothelial growth factor from an internal
ribosome entry site during hypoxia [19] It is worth noting
that upstream open reading frames and CAP-independent
translation are commonly found in genes whose products
play roles in the regulation of cell growth and in the cellular
response to stress
Interestingly, deregulation of translation initiation is a
common feature of tumorigenesis For example, increased
expression of the eukaryotic translation factor eIF4E has
been reported in a number of different cancers [21] It has
been speculated that overproduction of eIF4E in tumors
promotes translation of mRNAs with long and complex
5¢UTRs Indeed, sequence analysis demonstrated that
mRNAs with complex 5¢UTRs often encode for
proto-oncogenes [21] It remains to be shown whether E2F6 and/
or E2F6b play a role in tumorigenesis
A C K N O W L E D G E M E N T S
We wish to thankStefanie Hauser and our laboratory and divisional
colleagues for many helpful conversations We thankKelly Farrenkopf
for proofreading and for helpful comments We also thankGordon
Peters and William Kaelin for the E1B-luc and E2F1-luc constructs,
respectively This workwas supported by fellowships from the
Leukemia and Lymphoma Society and the Volkswagenstiftung to
S G.
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