mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells
Eukaryotic translation elongation factor 1A (eEF1A) exists as two 98 % homologous isoforms eEF1A1 and eEF1A2 that are tissue/development specific and differentially linked to apoptosis/cancerogenesis. A2 is overexpressed in a number of tumors while unusual expression of A1 is observed in injured mus...
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| Date: | 2013 |
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Інститут молекулярної біології і генетики НАН України
2013
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| Cite this: | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells / A.A. Vislovukh, N.L. Gralievska, M.G. Naumovets, B.S. Negrutskii, A.V. El'skaya // Вiopolymers and Cell. — 2013. — Т. 29, №. 5. — С. 389-394. — Бібліогр.: 32 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859827001730269184 |
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| author | Vislovukh, A.A. Gralievska, N.L. Naumovets, M.G. Negrutskii, B.S. El'skaya, A.V. |
| author_facet | Vislovukh, A.A. Gralievska, N.L. Naumovets, M.G. Negrutskii, B.S. El'skaya, A.V. |
| citation_txt | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells / A.A. Vislovukh, N.L. Gralievska, M.G. Naumovets, B.S. Negrutskii, A.V. El'skaya // Вiopolymers and Cell. — 2013. — Т. 29, №. 5. — С. 389-394. — Бібліогр.: 32 назв. — англ. |
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| container_title | Вiopolymers and Cell |
| description | Eukaryotic translation elongation factor 1A (eEF1A) exists as two 98 % homologous isoforms eEF1A1 and eEF1A2 that are tissue/development specific and differentially linked to apoptosis/cancerogenesis. A2 is overexpressed in a number of tumors while unusual expression of A1 is observed in injured muscles. To approach a possible mechanism underlying induced changes in the relative amounts of the isoforms we examined the intrinsic stability of the proteins and their mRNAs in human cancer cells. Aim. To estimate half-life of the isoforms of eEF1A at mRNA and protein level in human cancer cells. Methods. To measure mRNA stability the transcriptional block technique was applied, with subsequent analysis of the mRNA level by qPCR. To determine the protein decay rate the translation was blocked by cycloheximide and changes in the protein level were detected by Western blot. Results. Calculation of the protein stability revealed half-life of 72 for eEF1A1 and 95 hours for eEF1A2. Half-life of EEF1A1 and EEF1A2 mRNAs were 3 and 60 hours respectively. Conclusions. Despite similar protein stability, the isoforms of eEF1A dramatically differ in the half-lives of their mRNAs, suggesting that the mRNA decay mechanism is one of the main regulators of eEF1A1/A2 amount in MCF7 cancer cells.
Евкаріотний фактор елонгації трансляції (eEF1A) існує у вигляді двох гомологічних на 98 % ізоформ eEF1A1 і eEF1A2, які є тканиноспецифічними, відрізняються за представленістю в онтогенезі та по-різному пов’язані з апоптозом і канцерогенезом. Згідно з попередніми даними, eEF1A2 має підвищений рівень експресії у деяких пухлинах, а eEF1A1 – у пошкоджених м’язах. Щоб зрозуміти механізм, за яким змінюється відносна кількість ізоформ, ми дослідили стабільність білків та їхніх мРНК у клітинах раку людини. Мета. Оцінити час напівжиття ізоформ eEF1A на рівні мРНК і білка в клітинах раку людини. Методи. Для вимірювання стабільності мРНК використано техніку блокування транскрипції з подальшим аналізом рівня мРНК із застосуванням кількісної ПЛР. Для визначення швидкості розпаду білка трансляцію блокували циклогексимідом, подальші зміни рівня білка виявляли методом Вестерн-блоту. Результати. За підрахунками, стабільність білка зберігалася протягом 72 год у разі eEF1A1 та 95 год – у разі eEF1A2. Значення часу напівжиття EEF1A1 і EEF1A2 мРНК становлять відповідно 3 і 60 год. Висновки. Незважаючи на подібні значення стабільності білка, ізоформи eEF1A значно відрізняються за часом напівжиття їхніх мРНК, внаслідок чого можна припустити, що контроль стабільності мРНК є одним з основних механізмів регуляції експресії eEF1A1/ A2 в клітинах раку молочної залози MCF7.
Эукариотический фактор элонгации трансляции (eEF1A) существует в виде двух гомологичных на 98 % изоформ eEF1A1 и eEF1A2, являющихся тканеспецифическими, отличающихся представленностью в онтогенезе и по-разному связанных с апоптозом и канцерогенезом. Согласно наблюдениям, eEF1A2 имеет повышенный уровень экспрессии в некоторых опухолях, а eEF1A1 – в поврежденных мышцах. Чтобы понять механизм, по которому изменяется относительное количество изоформ, мы исследовали стабильность белков и их мРНК в клетках рака человека. Цель. Оценить время полужизни изоформ eEF1A на уровне мРНК и белка в клетках рака человека. Методы. Для измерения стабильности мРНК использовали технику блокирования транскрипции с последующим анализом уровня мРНК с применением количественной ПЦР. Для определения скорости распада белка трансляцию блокировали циклогексимидом, дальнейшие изменения уровня белка выявляли методом Вестерн-блота. Результаты. По расчетам, стабильность белка сохранялась в течение 72 ч в случае eEF1A1 и 95 ч – в случае eEF1A2. Время полужизни EEF1A1 и EEF1A2 мРНК составляло соответственно 3 и 60 ч. Выводы. Несмотря на подобные значения стабильности белка, изоформы eEF1A значительно отличаются по времени полужизни их мРНК, вследствие чего можно предположить, что контроль стабильности мРНК является одним из основных механизмов регуляции экспрессии eEF1A1/A2 в клетках рака молочной железы MCF7.
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| fulltext |
UDC 577.2
mRNAs coding for A1 and A2 isoforms of translation factor
eEF1 demonstrate different half-lives while A1 and A2
proteins are similarly stable in MCF7 cells
A. A. Vislovukh, N. L. Gralievska, M. G. Naumovets, B. S. Negrutskii, A. V. El’skaya
State Key Laboratory of Molecular and Cellular Biology
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
a.a.vislovukh@imbg.org.ua
Eukaryotic translation elongation factor 1A (eEF1A) exists as two 98 % homologous isoforms eEF1A1 and
eEF1A2 that are tissue/development specific and differentially linked to apoptosis/cancerogenesis. A2 is over-
expressed in a number of tumors while unusual expression of A1 is observed in injured muscles. To approach a
possible mechanism underlying induced changes in the relative amounts of the isoforms we examined the int-
rinsic stability of the proteins and their mRNAs in human cancer cells. Aim. To estimate half-life of the isoforms
of eEF1A at mRNA and protein level in human cancer cells. Methods. To measure mRNA stability the transcrip-
tional block technique was applied, with subsequent analysis of the mRNA level by qPCR. To determine the pro-
tein decay rate the translation was blocked by cycloheximide and changes in the protein level were detected by
Western blot. Results. Calculation of the protein stability revealed half-life of 72 for eEF1A1 and 95 hours for
eEF1A2. Half-life of EEF1A1 and EEF1A2 mRNAs were 3 and 60 hours respectively. Conclusions. Despite si-
milar protein stability, the isoforms of eEF1A dramatically differ in the half-lives of their mRNAs, suggesting that
the mRNA decay mechanism is one of the main regulators of eEF1A1/A2 amount in MCF7 cancer cells.
Keywords: eEF1A1, eEF1A2, eukaryotic translation elongation factor 1A, mRNA half-life, protein half-life.
Introduction. eEF1A is one of the main components of
translational apparatus that provides delivery of amino-
acylated tRNA to the A site of ribosome during elonga-
tion step of protein biosynthesis [1]. eEF1A exists as two
isoforms, eEF1A1 and eEF1A2. The reason behind the
appearance of two rather than one isoform of eEF1A in
higher vertebrates is unknown. During embryonic deve-
lopment eEF1A1 is expressed exclusively and ubiqui-
tously. However, throughout postnatal development in
cardiac, muscle and neuronal tissues a switch occurs from
the eEF1A1 to eEF1A2 expression. This change is cru-
cial, as the mice with a partial deletion of the EEF1A2
gene die on the 28th day after birth [2]. eEF1A1 and
eEF1A2 isoforms are 98 % homologous, so is not sur-
prising that their translation activity is similar [3]. How-
ever, these proteins are very different in non-canonical
functions, for instance, eEF1A1 stimulates apoptosis [4,
5] while eEF1A2 demonstrates anti-apoptotic proper-
ties [6]. Moreover, EEF1A2 is a putative proto-oncoge-
ne [7] over expressed in a variety of tumor tissues [8,
9]. As mentioned before, the expression of A1 and A2
in tissues is mutually exclusive. However there are exa-
mples of their induced co-expression under extreme si-
tuation like the muscle injury [10] or tumorigenesis [9,
11] suggesting special need in appearance of A1 or A2
isoform, respectively, during the background presence
of a counterpart protein. Specific non-canonical roles
of the isoforms and mechanisms controlling their res-
pective quantities in corresponding tissues are unknown.
First step towards elucidating these mechanisms is to
examine intrinsic stability of the corresponding mRNAs
and proteins. Breast cancer MCF7 cell line is selected
for this purpose as these cells demonstrate relatively
high expression level of eEF1A2.
389
ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 5. P. 389–394 doi: 10.7124/bc.00082E
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013
Materials and methods. Cell line, cycloheximide
and actinomycin treatment. MCF7 cells were cultured in
DMEM («Sigma», USA) growth medium with stabili-
zed L-glutamine, contained 10 % FBS («Sigma») and
1 % Penicillin/Streptomycin («Sigma»). Cells were ma-
intained at 37 °C in a humidified atmosphere containing
5 % CO2. To determine mRNAs half-lives, MCF7 cells
were treated with 5 µg/ml actinomycin D («Sigma»).
Cells were harvested at the time points indicated in the
Figures. Amount of EEF1A1/A2 mRNAs were quan-
tified by qPCR. For protein half-life determination,
MCF7 cells were incubated with 100 µg/ml cyclohexi-
mide («Sigma»). Cells were harvested at the time points
indicated in the Figures with consequent Western blot
analyses. The Western blot results were quantified by
the ChemiDoc system («Bio-Rad», USA) Changes in
amount of eEF1A1, eEF1A2 and GAPDH proteins we-
re analysed by Gel Doc software («Bio-Rad»).
qPCR. Total RNA was isolated using TRI Reagent
(«Sigma»). 1 µg of RNA was used for cDNA synthesis
with a RevertAid Premium Reverse Transcriptase («Ther-
mo Scientific», USA) according to manufacturer recom-
mendations. Each reaction was performed in a mix of
20 µl reaction mixture containing 1 µl cDNA, Maxima
SYBR Green qPCR Master Mix (2�) («Thermo Scien-
tific») and forward/reverse primers 0.3 µM each. The
primers for eEF1A2 qPCR and cycling conditions were
described earlier [10]. Primers for beta-actin were:
forward primer – 5'-GCGGGAAATCGTGCGTGAC
ATT-3'; reverse primer – 5'- GATGGAGTTGAAGGT
AGTTTCGTG-3'. QPCR was quantified with MyiQ
real-time PCR system («Bio-Rad»). Data were analy-
zed using qPCR Miner 4.0 software [12].
Western blot. Total cell lysates were prepared in M-
PER buffer («Pierce», USA) supplemented with the pro-
tease inhibitor cocktail («Roche», France). Cells were
incubated at 4 °C for 20 min. Lysates were centrifuged
and supernatant was stored in liquid nitrogen. Proteins
were separated by PAGE 10 % (29:1) («Bio-Rad») and
transferred to PVDF membranes («Millipore», USA).
Membranes were developed using Immobi lon Western
Chemiluminicsent Substrate («Millipore»).
Results and Discussion. As eEF1A2 is a putative
proto-oncogene and such proteins usually are quickly
degraded [13] we expected the life time of eEF1A2 to
be significantly less than eEF1A1. There were several
datasheets on the eEF1A1 and eEF1A2 half-lives ob-
tained from global proteomic studies examining a vast
majority of cellular proteins at once [14–16]. Unfortu-
nately, these data did not give a definite answer, even in
the case the same cell line was used in different studies
(Table). To clarify the issue we measured half-lives of
eEF1A1 and eEF1A2 directly, using cycloheximi-
de-treated MCF-7 cells and two kinds of antibodies, the
first recognizing both A1 and A2 isoforms and the se-
cond being exclusively specific for A2 [17]. As the
amount of A1 in MCF7 cells is much larger (by orders)
than A2 we consider the former antibodies response re-
flects mostly the A1 amount. Cycloheximide is an anti-
biotic which specifically blocks elongation step of pro-
tein biosynthesis [18]. That is why it is often used for
protein stability measurements [19].
Surprisingly, the eEF1A1 and eEF1A2 isoforms
demonstrated similar decay rates (Fig. 1, A). Subsequ-
ent calculations revealed that the both isoforms belong
to the class of long lived proteins with estimated half-
lives of 72 for eEF1A1 and 95 hours for eEF1A2 (Fig.
1, B). GAPDH which is a known long-lived protein was
planned to be used as a loading control. However, as
the half-life of GADPH (61 h) was found to be com-
parable with that for eEF1A1 and eEF1A2 (Fig. 1, A)
we had to use strictly similar amount of cells for prepa-
ring the extract to load per line. Thus, both eEF1A1 and
eEF1A2 isoforms possess comparable half-lives in the
breast cancer MCF-7 cells. We suggest that protein sta-
bilization does not seem to contribute to cancer-related
change in the intracellular eEF1A1/eEF1A2 ratio and
the alterations in relative amount of eEF1A1 and eEF1A2
in cancer cells are governed by the level and translation
efficacy of their mRNAs.
390
VISLOVUKH A. A. ET AL.
Protein
name
Half-lives of proteins, h
HeLa [14] HeLa [16] NIH 3T3 [15] C2C12 [14]
eEF1A1 75 56,9 126 82,3
eEF1A2
Too high for
detection
83,5 103,16 NA
GAPDH
Too high for
detection
68,4 353,55 94,9
Comparison of eEF1A2, eEF1A2 and GAPDH proteins half-lives
obtained by different studies
We examined the parameter of mRNA stability by
comparing the decay rates of the EEF1A1 and EEF1A2
mRNAs. mRNAs coding for the isoforms are highly
homologous in open reading frames (ORF), however,
they are rather different in the 3' and 5' untranslated
regions (UTRs), which may provide a background for
specific posttranscriptional control. The presence of
such structural elements as CPE (cytoplasmic polyade-
nylation element), ARE (AU-rich element) etc. in the
EEF1A1 rather than in the EEF1A2 mRNA suggests
lower stability of the former. Varieties of methods were
developed to quantify the decay rates of mRNA in dif-
ferent cell lines. The most exploited is «transcriptional
shut off» method to block transcription by RNA poly-
merase inhibitors, with subsequent measurement of the
residual amounts of mRNAs of the interest by Northern
Blot or qPCR.
Therefore, MCF7 cells were treated with actinomy-
cin D to block RNA polymerase II activity. Cells were
collected after 2, 4, 8, 28, 48 and 80 h of incubation
with consequent analysis of the EEF1A1/1A2 mRNA
level by qPCR. Indeed, mRNA of eEF1A2 was found
to be considerably more stable than eEF1A1 (Fig. 2).
Approximate half-life times were 60 h for eEF1A2 and
3 h for eEF1A1 mRNAs. In the literature, there is the on-
ly indication of half-life measurement for the eEF1A1
mRNA [15] where Schwanhausser et al. observed that
half-life of the eEF1A1 mRNA is 17,5 h. However, the
authors measured mRNA decay rates in non-cancerous
mouse fibroblasts (NIH3T3), while our experiments we-
re carried out in MCF7 human cancer cells. Consequ-
ently such divergence in the results can be partly explai-
ned by the difference in 3' UTRs of human and mouse
EEF1A1 mRNAs [20]. 3' UTR of the human EEF1A1
mRNA is much longer and contains several additional in-
stability elements. Besides, rate of the EEF1A1 mRNA
decay in cancer and non-cancerous cells may be different.
It is known that transcription of the EEF1A1 mRNA
occurs very powerfully, the promoter region of eef1a
gene is the second most efficient after cytomegalovirus
one [21]. Moreover the EEF1A1 mRNA belongs to the
TOP class of mRNAs [22] and consequently A1 can be
rapidly synthesized in response to the growth stimuli. It
should be mentioned that the EEF1A2 mRNA also con-
tains a non-canonical TOP element [23], however its
functionality is still questionable. We discover now that
to prevent over saturation of a cell with rather stable pro-
tein eEF1A1 a novel mechanism was elaborated which
includes rapid degradation of EEF1A1 mRNA soon af-
ter its synthesis. Thus, repression of transcription of the
eEF1A1 mRNA appears to be though not very fast but
reliable way to decrease the amount of eEF1A1 protein
with subsequent inhibition of translation elongation. The
proto-oncogenic eEF1A2 protein may be much less cont-
rolled by means of down-regulation of transcription or
translation. Interestingly, eEF1A2 is exclusively expres-
sed in such very important cells as myocytes and neu-
rons [3, 24]. Virtual absence of the possibility to quick-
ly alter its amount may provide additional reliability and
stability of the protein synthesis in these terminally dif-
391
mRNAs CODING FOR A1 AND A2 ISOFORMS OF TRANSLATION FACTOR eEF1A
eEF1A1
eEF1A2
GAPDH
0 2 4 8 28 48 80
P
er
ce
n
ts
o
f
re
m
a
in
in
g
p
ro
te
in
Time, h
0 20 40 60 80
40
60
80
100
120
140
160
2
1
3
A B
Fig. 1. Estimation of eEF1A1 and eEF1A2 half-lives in MCF7 cells: A – total protein biosynthesis was blocked by cycloheximide (at the indicated
time points, 3 � 10
6
cells were collected and analysed by Western blot with antibodies against A1/A2 and A2 proteins); B – representation of the
data in semi-log plot: 1 – eEF1A1; 2 – eEF1A2; 3 – GAPDH (data were fitted by exponential decay first order kinetic function; t
1/2
= ln2/k, where k
is the rate constant for protein decay)
ferentiated cells. On the other hand, over expression of
eEF1A2 could be very harmful for cells and lead to on-
cogenesis [8]. To control the amount of eEF1A2, a cell
has elaborated another mechanism, exactly down-regu-
lation of eEF1A2 by microRNA-663 and 744 recently
described by us [25]. The organism seems to keep the
eEF1A2 expression under a tight control, limiting its ap-
pearance in terminally differentiated cells. On the cont-
rary, eEF1A1 is normally found everywhere in the orga-
nism except muscles and neurons. The eEF1A1 mRNA,
opposite to A2, has short, TOP class 5' UTR and long 3'
UTR which opens wide «place d’arme» to the trans-
factors [20, 26]. Consequently, this mRNA has much
shorter half-life, that also contributes to the regulatory
process.
For instance, the neurons respond to electric stimuli
via certain parts of the dendrites rather than via whole
soma [27]. In that case, local protein biosynthesis and/or
cytoskeleton reorganization should be activated to afford
neuronal plasticity [28]. We assume that the eEF1A2
mRNA being stable may provide homeostasis and stabi-
lity of the soma, while the EEF1A1 mRNA could be res-
ponsible for the fast structural changes via cytoskeleton
reorganization and local protein biosynthesis in distal
compartments of neuron. It is already known that upon
LTP (long term potentiation) formation in such distal
compartments as dendrites, mTOR-induced translation
of the eEF1A1 mRNA is observed [29–31]. Another
example is muscles, where the isoform eEF1A2 is the
only one, while the induction of EEF1A1 expression is
observed during regeneration processes after muscle
injury [10]. After healing is finished the eEF1A1 mRNA
can degrade quickly because of low half-life, permit-
ting to restore the normal functioning. Interestingly,
muscles healing process largely recapitulates embryo-
nic myogenesis where the only eEF1A1 isoform is pre-
sent [32].
Besides, high level of homology and similar cataly-
tic function during protein biosynthesis, the isoforms
of eEF1A are very different in their numerous non-ca-
nonical functions.
Combining our and literature data we put forward a
novel concept stating that the existence of the two high-
ly homologous isoforms of human eEF1A can be explai-
ned by the necessity of cells to control selectively ex-
pression of the eEF1A1/A2 isoforms at different levels
and by different mechanisms depending on their cano-
nical and non-canonical functions.
eEF1A1 is constitutively expressed in majority of
the organs in the organism providing a basic level of
protein synthesis responsive to different kinds of regula-
tion. Appearance of eEF1A2 in the same tissues seems
not to respond to the same regulatory factors, therefore,
may be very harmful for cells.
Conclusions. The data show that the isoforms of
eEF1A demonstrate very different half-lives of mRNA
but similar stability of corresponding proteins. We assu-
me that the mRNA decay mechanism is one of the main
regulators of eEF1A1 expression, at least, in the human
breast cancer MCF7 cells.
À. À. ³ñëîâóõ, Í. Ë. Ãðàëºâñüêà, Ì. Ã. Íàóìîâåöü,
Á. Ñ. Íåãðóöüêèé, À. Â. ªëüñüêà
ìÐÍÊ, ùî êîäóþòü A1 ³ A2 ³çîôîðìè ôàêòîðà òðàíñëÿö³¿ eEF1, ìà-
þòü ð³çíèé ÷àñ íàï³âæèòòÿ, ó òîé ÷àñ ÿê á³ëêè À1 ³ À2 ð³âíîþ
ì³ðîþ ñòàá³ëüí³ â êë³òèíàõ MCF7
Ðåçþìå
Åâêàð³îòíèé ôàêòîð åëîíãàö³¿ òðàíñëÿö³¿ (eEF1A) ³ñíóº ó âèãëÿä³
äâîõ ãîìîëîã³÷íèõ íà 98 % ³çîôîðì eEF1A1 ³ eEF1A2, ÿê³ º òêàíè-
íîñïåöèô³÷íèìè, â³äð³çíÿþòüñÿ çà ïðåäñòàâëåí³ñòþ â îíòîãåíåç³
òà ïî-ð³çíîìó ïîâ’ÿçàí³ ç àïîïòîçîì ³ êàíöåðîãåíåçîì. Çã³äíî ç
ïîïåðåäí³ìè äàíèìè, eEF1A2 ìຠï³äâèùåíèé ð³âåíü åêñïðåñ³¿ ó
äåÿêèõ ïóõëèíàõ, à eEF1A1 – ó ïîøêîäæåíèõ ì’ÿçàõ. Ùîá çðî-
çóì³òè ìåõàí³çì, çà ÿêèì çì³íþºòüñÿ â³äíîñíà ê³ëüê³ñòü ³çîôîðì,
ìè äîñë³äèëè ñòàá³ëüí³ñòü á³ëê³â òà ¿õí³õ ìÐÍÊ ó êë³òèíàõ ðàêó
ëþäèíè. Ìåòà. Îö³íèòè ÷àñ íàï³âæèòòÿ ³çîôîðì eEF1A íà ð³âí³
ìÐÍÊ ³ á³ëêà â êë³òèíàõ ðàêó ëþäèíè. Ìåòîäè. Äëÿ âèì³ðþâàííÿ
ñòàá³ëüíîñò³ ìÐÍÊ âèêîðèñòàíî òåõí³êó áëîêóâàííÿ òðàíñêðèï-
392
VISLOVUKH A. A. ET AL.
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1
2
Fig. 2. Estimation of EEF1A1 (1) and EEF1A2 (2) mRNAs stability in
MCF7 cells. Transcription was blocked by actinomycin D. At the time
points indicated, the samples were collected and qPCR analyses of
EEF1A1/A2 mRNAs was carried out. Data were fitted by exponential
decay first order kinetic function; t
1/2
= ln2/k, where k is the rate
constant for mRNA decay
ö³¿ ç ïîäàëüøèì àíàë³çîì ð³âíÿ ìÐÍÊ ³ç çàñòîñóâàííÿì ê³ëüê³ñíî¿
ÏËÐ. Äëÿ âèçíà÷åííÿ øâèäêîñò³ ðîçïàäó á³ëêà òðàíñëÿö³þ áëîêó-
âàëè öèêëîãåêñèì³äîì, ïîäàëüø³ çì³íè ð³âíÿ á³ëêà âèÿâëÿëè ìåòî-
äîì Âåñòåðí-áëîòó. Ðåçóëüòàòè. Çà ï³äðàõóíêàìè, ñòàá³ëüí³ñòü
á³ëêà çáåð³ãàëàñÿ ïðîòÿãîì 72 ãîä ó ðàç³ eEF1A1 òà 95 ãîä – ó ðàç³
eEF1A2. Çíà÷åííÿ ÷àñó íàï³âæèòòÿ EEF1A1 ³ EEF1A2 ìÐÍÊ
ñòàíîâëÿòü â³äïîâ³äíî 3 ³ 60 ãîä. Âèñíîâêè. Íåçâàæàþ÷è íà ïî-
ä³áí³ çíà÷åííÿ ñòàá³ëüíîñò³ á³ëêà, ³çîôîðìè eEF1A çíà÷íî â³äð³ç-
íÿþòüñÿ çà ÷àñîì íàï³âæèòòÿ ¿õí³õ ìÐÍÊ, âíàñë³äîê ÷îãî ìîæ-
íà ïðèïóñòèòè, ùî êîíòðîëü ñòàá³ëüíîñò³ ìÐÍÊ º îäíèì ç îñ-
íîâíèõ ìåõàí³çì³â ðåãóëÿö³¿ åêñïðåñ³¿ eEF1A1/ A2 â êë³òèíàõ ðàêó
ìîëî÷íî¿ çàëîçè MCF7.
Êëþ÷îâ³ ñëîâà: eEF1A1, eEF1A2, åâêàð³îòíèé ôàêòîð åëîíãà-
ö³¿ òðàíñëÿö³¿ 1À, ÷àñ íàï³âæèòòÿ ìÐÍÊ, ÷àñ íàï³âæèòòÿ á³ëêà.
À. À. Âèñëîâóõ, Í. Ë. Ãðàëåâñêàÿ, Ì. Ã. Íàóìîâåö, Á. Ñ. Íåãðóöêèé,
À. Â. Åëüñêàÿ
ìÐÍÊ, êîäèðóþùèå À1 è À2 èçîôîðìû ôàêòîðà òðàíñëÿöèè
eEF1, èìåþò ðàçëè÷íîå âðåìÿ ïîëóæèçíè, à áåëêè À1 è À2
â ðàâíîé ñòåïåíè ñòàáèëüíû â êëåòêàõ MCF7
Ðåçþìå
Ýóêàðèîòè÷åñêèé ôàêòîð ýëîíãàöèè òðàíñëÿöèè (eEF1A) ñóùå-
ñòâóåò â âèäå äâóõ ãîìîëîãè÷íûõ íà 98 % èçîôîðì eEF1A1 è
eEF1A2, ÿâëÿþùèõñÿ òêàíåñïåöèôè÷åñêèìè, îòëè÷àþùèõñÿ ïðåä-
ñòàâëåííîñòüþ â îíòîãåíåçå è ïî-ðàçíîìó ñâÿçàííûõ ñ àïîïòî-
çîì è êàíöåðîãåíåçîì. Ñîãëàñíî íàáëþäåíèÿì, eEF1A2 èìååò ïî-
âûøåííûé óðîâåíü ýêñïðåññèè â íåêîòîðûõ îïóõîëÿõ, à eEF1A1 –
â ïîâðåæäåííûõ ìûøöàõ. ×òîáû ïîíÿòü ìåõàíèçì, ïî êîòîðîìó
èçìåíÿåòñÿ îòíîñèòåëüíîå êîëè÷åñòâî èçîôîðì, ìû èññëåäîâà-
ëè ñòàáèëüíîñòü áåëêîâ è èõ ìÐÍÊ â êëåòêàõ ðàêà ÷åëîâåêà. Öåëü.
Îöåíèòü âðåìÿ ïîëóæèçíè èçîôîðì eEF1A íà óðîâíå ìÐÍÊ è áåë-
êà â êëåòêàõ ðàêà ÷åëîâåêà. Ìåòîäû. Äëÿ èçìåðåíèÿ ñòàáèëüíî-
ñòè ìÐÍÊ èñïîëüçîâàëè òåõíèêó áëîêèðîâàíèÿ òðàíñêðèïöèè ñ
ïîñëåäóþùèì àíàëèçîì óðîâíÿ ìÐÍÊ ñ ïðèìåíåíèåì êîëè÷åñò-
âåííîé ÏÖÐ. Äëÿ îïðåäåëåíèÿ ñêîðîñòè ðàñïàäà áåëêà òðàíñëÿ-
öèþ áëîêèðîâàëè öèêëîãåêñèìèäîì, äàëüíåéøèå èçìåíåíèÿ óðîâíÿ
áåëêà âûÿâëÿëè ìåòîäîì Âåñòåðí-áëîòà. Ðåçóëüòàòû. Ïî ðàñ-
÷åòàì, ñòàáèëüíîñòü áåëêà ñîõðàíÿëàñü â òå÷åíèå 72 ÷ â ñëó÷àå
eEF1A1 è 95 ÷ – â ñëó÷àå eEF1A2. Âðåìÿ ïîëóæèçíè EEF1A1 è
EEF1A2 ìÐÍÊ ñîñòàâëÿëî ñîîòâåòñòâåííî 3 è 60 ÷. Âûâîäû. Íå-
ñìîòðÿ íà ïîäîáíûå çíà÷åíèÿ ñòàáèëüíîñòè áåëêà, èçîôîðìû
eEF1A çíà÷èòåëüíî îòëè÷àþòñÿ ïî âðåìåíè ïîëóæèçíè èõ ìÐÍÊ,
âñëåäñòâèå ÷åãî ìîæíî ïðåäïîëîæèòü, ÷òî êîíòðîëü ñòàáèëü-
íîñòè ìÐÍÊ ÿâëÿåòñÿ îäíèì èç îñíîâíûõ ìåõàíèçìîâ ðåãóëÿöèè
ýêñïðåññèè eEF1A1/A2 â êëåòêàõ ðàêà ìîëî÷íîé æåëåçû MCF7.
Êëþ÷åâûå ñëîâà: eEF1A1, eEF1A2, ýóêàðèîòè÷åñêèé ôàêòîð
ýëîíãàöèè òðàíñëÿöèè 1À, âðåìÿ ïîëóæèçíè ìÐÍÊ, âðåìÿ ïîëó-
æèçíè áåëêà.
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VISLOVUKH A. A. ET AL.
|
| id | nasplib_isofts_kiev_ua-123456789-153178 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T15:29:34Z |
| publishDate | 2013 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Vislovukh, A.A. Gralievska, N.L. Naumovets, M.G. Negrutskii, B.S. El'skaya, A.V. 2019-06-13T15:17:32Z 2019-06-13T15:17:32Z 2013 mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells / A.A. Vislovukh, N.L. Gralievska, M.G. Naumovets, B.S. Negrutskii, A.V. El'skaya // Вiopolymers and Cell. — 2013. — Т. 29, №. 5. — С. 389-394. — Бібліогр.: 32 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00082E https://nasplib.isofts.kiev.ua/handle/123456789/153178 577.2 Eukaryotic translation elongation factor 1A (eEF1A) exists as two 98 % homologous isoforms eEF1A1 and eEF1A2 that are tissue/development specific and differentially linked to apoptosis/cancerogenesis. A2 is overexpressed in a number of tumors while unusual expression of A1 is observed in injured muscles. To approach a possible mechanism underlying induced changes in the relative amounts of the isoforms we examined the intrinsic stability of the proteins and their mRNAs in human cancer cells. Aim. To estimate half-life of the isoforms of eEF1A at mRNA and protein level in human cancer cells. Methods. To measure mRNA stability the transcriptional block technique was applied, with subsequent analysis of the mRNA level by qPCR. To determine the protein decay rate the translation was blocked by cycloheximide and changes in the protein level were detected by Western blot. Results. Calculation of the protein stability revealed half-life of 72 for eEF1A1 and 95 hours for eEF1A2. Half-life of EEF1A1 and EEF1A2 mRNAs were 3 and 60 hours respectively. Conclusions. Despite similar protein stability, the isoforms of eEF1A dramatically differ in the half-lives of their mRNAs, suggesting that the mRNA decay mechanism is one of the main regulators of eEF1A1/A2 amount in MCF7 cancer cells. Евкаріотний фактор елонгації трансляції (eEF1A) існує у вигляді двох гомологічних на 98 % ізоформ eEF1A1 і eEF1A2, які є тканиноспецифічними, відрізняються за представленістю в онтогенезі та по-різному пов’язані з апоптозом і канцерогенезом. Згідно з попередніми даними, eEF1A2 має підвищений рівень експресії у деяких пухлинах, а eEF1A1 – у пошкоджених м’язах. Щоб зрозуміти механізм, за яким змінюється відносна кількість ізоформ, ми дослідили стабільність білків та їхніх мРНК у клітинах раку людини. Мета. Оцінити час напівжиття ізоформ eEF1A на рівні мРНК і білка в клітинах раку людини. Методи. Для вимірювання стабільності мРНК використано техніку блокування транскрипції з подальшим аналізом рівня мРНК із застосуванням кількісної ПЛР. Для визначення швидкості розпаду білка трансляцію блокували циклогексимідом, подальші зміни рівня білка виявляли методом Вестерн-блоту. Результати. За підрахунками, стабільність білка зберігалася протягом 72 год у разі eEF1A1 та 95 год – у разі eEF1A2. Значення часу напівжиття EEF1A1 і EEF1A2 мРНК становлять відповідно 3 і 60 год. Висновки. Незважаючи на подібні значення стабільності білка, ізоформи eEF1A значно відрізняються за часом напівжиття їхніх мРНК, внаслідок чого можна припустити, що контроль стабільності мРНК є одним з основних механізмів регуляції експресії eEF1A1/ A2 в клітинах раку молочної залози MCF7. Эукариотический фактор элонгации трансляции (eEF1A) существует в виде двух гомологичных на 98 % изоформ eEF1A1 и eEF1A2, являющихся тканеспецифическими, отличающихся представленностью в онтогенезе и по-разному связанных с апоптозом и канцерогенезом. Согласно наблюдениям, eEF1A2 имеет повышенный уровень экспрессии в некоторых опухолях, а eEF1A1 – в поврежденных мышцах. Чтобы понять механизм, по которому изменяется относительное количество изоформ, мы исследовали стабильность белков и их мРНК в клетках рака человека. Цель. Оценить время полужизни изоформ eEF1A на уровне мРНК и белка в клетках рака человека. Методы. Для измерения стабильности мРНК использовали технику блокирования транскрипции с последующим анализом уровня мРНК с применением количественной ПЦР. Для определения скорости распада белка трансляцию блокировали циклогексимидом, дальнейшие изменения уровня белка выявляли методом Вестерн-блота. Результаты. По расчетам, стабильность белка сохранялась в течение 72 ч в случае eEF1A1 и 95 ч – в случае eEF1A2. Время полужизни EEF1A1 и EEF1A2 мРНК составляло соответственно 3 и 60 ч. Выводы. Несмотря на подобные значения стабильности белка, изоформы eEF1A значительно отличаются по времени полужизни их мРНК, вследствие чего можно предположить, что контроль стабильности мРНК является одним из основных механизмов регуляции экспрессии eEF1A1/A2 в клетках рака молочной железы MCF7. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells мРНК, що кодують A1 і A2 ізоформи фактора трансляції eEF1A, мають різний час напівжиття, у той час як білки А1 і А2 рівною мірою стабільні в клітинах MCF7 мРНК, кодирующие А1 и А2 изоформы фактора трансляции eEF1А, имеют различное время полужизни, а белки А1 и А2 в равной степени стабильны в клетках MCF7 Article published earlier |
| spellingShingle | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells Vislovukh, A.A. Gralievska, N.L. Naumovets, M.G. Negrutskii, B.S. El'skaya, A.V. Structure and Function of Biopolymers |
| title | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells |
| title_alt | мРНК, що кодують A1 і A2 ізоформи фактора трансляції eEF1A, мають різний час напівжиття, у той час як білки А1 і А2 рівною мірою стабільні в клітинах MCF7 мРНК, кодирующие А1 и А2 изоформы фактора трансляции eEF1А, имеют различное время полужизни, а белки А1 и А2 в равной степени стабильны в клетках MCF7 |
| title_full | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells |
| title_fullStr | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells |
| title_full_unstemmed | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells |
| title_short | mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells |
| title_sort | mrnas coding for a1 and a2 isoforms of translation factor eef1a demonstrate different half-lives while a1 and a2 proteins are similarly stable in mcf7 cells |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/153178 |
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