Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform
Aim. To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone. Methods. RT-PCR, DNA sequencing, Western blotting. Results. RT-PCR analysis of total RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1 splice variant that possesses additional ex...
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| Опубліковано в: : | Вiopolymers and Cell |
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| Дата: | 2019 |
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| Формат: | Стаття |
| Мова: | Англійська |
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Інститут молекулярної біології і генетики НАН України
2019
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform / I.V. Zaiets, V.V. Holiar, V.V. Filonenko // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 99-106. — Бібліогр.: 24 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859879110551011328 |
|---|---|
| author | Zaiets, I.V. Holiar, V.V. Filonenko, V.V. |
| author_facet | Zaiets, I.V. Holiar, V.V. Filonenko, V.V. |
| citation_txt | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform / I.V. Zaiets, V.V. Holiar, V.V. Filonenko // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 99-106. — Бібліогр.: 24 назв. — англ. |
| collection | DSpace DC |
| container_title | Вiopolymers and Cell |
| description | Aim. To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone. Methods. RT-PCR, DNA sequencing, Western blotting. Results. RT-PCR analysis of total RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1 splice variant that possesses additional exon 1a and encodes the p60 isoform of S6K1 only. Moreover, RT-PCR and western blotting were applied to estimate the expression levels of the p60-S6K1 mRNA and protein. A heterogeneous expression of the p60-S6K1 transcript was observed; it did not correlate with the total S6K1 mRNA expression and with the protein content of p60-S6K1 in the studied cell lines. Conclusions. We provide the evidence for the existence of a novel S6K1 splice variant coding for the p60-S6K1 isoform. The cell can employ two distinct pathways of the p60-S6K1 expression based on alternative translation of mRNA common for the main S6K1 isoforms mRNA and/or translation of the novel p60-S6K1 specific mRNA. Since the levels of the p60-S6K1 transcript expression do not correlate with the expression of total S6K1 mRNA, it is plausible that the p60-S6K1 isoform plays a role distinct from that of other isoforms in cellular physiology, as well as in the development of S6K1-related pathologies.
Мета. З'ясувати можливість існування сплайсової мРНК кінази S6К1 специфічної виключно для р60-S6К1 ізоформи. Методи. ЗТ-ПЛР, ДНК секвенування, Вестерн-блоттинг. Результати. ЗТ-ПЛР аналіз тотальної РНК, виділеної із клітин лінії MCF-7, з наступним секвенуванням ДНК виявив новий сплайсовий варіант мРНК S6K1, що містить додатковий екзон 1а і кодує лише p60 ізоформу S6K1. Для оцінки рівня експресії p60-S6K1 мРНК та білку було застосовано ЗТ-ПЛР та вестерн-блот. Результати. вказують на гетерогенну експресію транскрипту p60-S6K1, що не корелює ні з експресією загальної S6K1 мРНК, ні з білковим вмістом p60-S6K1 у досліджуваних клітинних лініях. Висновки. Надано докази існування нової сплайсової мРНК S6K1, яка кодує ізоформу p60-S6K1, що свідчить про існування в клітині двох незалежних шляхів експресії p60-S6K1, а саме альтернативну трансляцію спільної для головних S6K1 ізоформ мРНК та/чи трансляцію нової специфічної лише для р60-S6K1 сплайсової мРНК. Оскільки рівень експресії p60-S6K1 транскрипту в різних клітинних лініях не корелює із експресією загальної мРНК S6K1, цілком можливо, що p60-S6K1 ізоформа може відігравати відмінну від інших S6K1 ізоформ роль у клітинній фізіології, а також при розвитку патологій, які пов’язані із S6K1.
Цель. Выяснить возможность существования сплайсинговой мРНК киназы S6К1 специфической исключительно для р60-S6К1 изоформы. Методы. ОТ-ПЦР, ДНК секвенирование, Вестерн-блоттинг. Результаты. ОТ-ПЦР анализ тотальной РНК, выделенной из клеток линии MCF-7, с последующим секвенированием ДНК выявил новый сплайсовый вариант S6K1, содержит дополнительный экзон 1а и кодирует только изоформу p60-S6K1. Далее были использованы ОТ-ПЦР и вестерн-блоттинг для оценки уровней p60-S6K1 мРНК и белка. Результаты показали гетерогенную экспрессию транскрипта p60-S6K1, экспрессия которого не коррелировала ни с тотальной S6K1 мРНК, ни с белковым содержанием p60-S6K1 в исследуемых клеточных линиях. Выводы. Предоставлены доказательства существования нового сплайсового варианта S6K1, который кодирует изоформу p60-S6K1, что указывает на существование в клетке двух независимых путей экспрессии p60-S6K1 изоформы, а именно альтернативную трансляцию общей для основных изоформ мРНК и/или трансляцию новой специфичной лишь для p60-S6K1 сплайсовой мРНК. Поскольку уровень экспрессии p60-S6K1 транскрипта в различных клеточных линиях не коррелируют с экспрессией тотальной S6K1 мРНК, можно предположить, что p60-S6K1 изоформа может играть отличную то других S6K1 изоформ роль в клеточной физиологии, а также при развитии патологий, которые связаны с S6K1.
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I. V. Zaiets, V. V. Holiar, V. V. Filonenko
© 2019 I. V. Zaiets et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
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provided the original work is properly cited
UDC 577
Identification of a novel S6K1 splice variant coding
for the p60-S6K1 isoform
I. V. Zaiets, V. V. Holiar, V. V. Filonenko
Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnogo str., 03680, Kyiv, Ukraine
filonenko@imbg.org.ua
Aim. To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone.
Methods. RT-PCR, DNA sequencing, Western blotting. Results. RT-PCR analysis of total
RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1
splice variant that possesses additional exon 1a and encodes the p60 isoform of S6K1 only.
Moreover, RT-PCR and western blotting were applied to estimate the expression levels of the
p60-S6K1 mRNA and protein. A heterogeneous expression of the p60-S6K1 transcript was
observed; it did not correlate with the total S6K1 mRNA expression and with the protein
content of p60-S6K1 in the studied cell lines. Conclusions. We provide the evidence for the
existence of a novel S6K1 splice variant coding for the p60-S6K1 isoform. The cell can employ
two distinct pathways of the p60-S6K1 expression based on alternative translation of mRNA
common for the main S6K1 isoforms mRNA and/or translation of the novel p60-S6K1 spe-
cific mRNA. Since the levels of the p60-S6K1 transcript expression do not correlate with the
expression of total S6K1 mRNA, it is plausible that the p60-S6K1 isoform plays a role distinct
from that of other isoforms in cellular physiology, as well as in the development of S6K1-
related pathologies.
K e y w o r d s: p60-S6K1 transcript, alternative splicing, gene expression
Introduction
The ribosomal protein S6 kinase 1 (S6K1,
RPS6KB1 gene) plays an important role in pro-
moting fundamental cellular functions, including
cell metabolism, growth and motility, and repre-
sents a downstream effector of the mTORC1
signaling pathway [1]. Indeed, the aberrant
mTORC1 signaling underlies a number of path-
ological conditions [2]. Numerous data indicate
the implication of S6K1 in the development of
human cancer. A pathological cellular response
in cancer is frequently associated with amplifica-
tion and overexpression of the S6K1 gene [3–19].
Current data suggest that most effects of S6K1
are mediated by the major p70 and p85 isoforms
that are originated from alternative mRNA trans-
lation [1]. Little attention, however, has been paid
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2019. Vol. 35. N 2. P 99–106
doi: http://dx.doi.org/10.7124/bc.00099B
mailto:filonenko@imbg.org.ua
100
I. V. Zaiets, V. V. Holiar, V. V. Filonenko
to studying the other isoforms. Recent data dem-
onstrate the ability of short S6K1 isoforms to
initiate tumorigenesis [20, 21], thus underscoring
important roles for these isoforms in cancer.
Additionally, an evidence exists for the p60-
S6K1 protein isoform [22]. It was assumed that
this isoform originates from an alternative trans-
lation event [22]. Moreover,our recent work has
confirmed such an assumption [23]. On the oth-
er hand, the analysis of current nucleic acid da-
tabases has revealed an additional S6K1 splice
variant (NM_001272044.1, RefSeq, NCBI) dis-
covered by NEDO human cDNA sequencing
project [unpublished data] that presumably could
be translated to the p60-S6K1 protein.
The main task of the current study was to
confirm the existence of mRNA coding for the
p60 isoform of S6K1 and to estimate its expres-
sion in various cell lines. Initially, we used RT-
PCR to identify the p60-S6K1 mRNA in breast
cancer cell line MCF-7. After that, DNA sequenc-
ing of the PCR product confirmed the identity of
the p60-S6K1 transcript which contains an inser-
tion (we called exon 1a) arising from intron 1. To
evaluate the expression le vels of the p60-S6K1
mRNA in cell lines we also applied RT-PCR. The
obtained data revealed heterogeneous expression
of the p60-S6K1 mRNA in the studied cell lines,
and this expression did not correlate with the
expression of total S6K1 mRNA. Addtionally, we
compared the levels of p60-S6K1 mRNA and
protein expression, demonstrating the absence of
correlation between them.
Materials and Methods
Cell culture. HEK-293, MCF-7 and HeLa, cell
cultures were maintained in Dulbecco’s modi-
fied Eagle medium (DMEM) (Gibco) supple-
mented with 10 % fetal bovine serum (Gibco),
100 units/ml penicillin, 100 µg/ml streptomycin,
and 2mM glutamine. Jurkat and U-937, and
HepG2, U-373 and U-87 cell lines were cul-
tured in RPMI-1640 (HyClone) and Eagle’s
minimum essential medium (EMEM) (Gibco),
respectively, containing supplements listed
above. All the cells used in this study were
grown in 6-cm cell culture dishes until they
reached 90 % confluency and lysed for subse-
quent total RNA isolation. Each cell culture was
maintained in a 5 % CO2 incubator at 37°C.
Oligonucleotides. For PCR detection of the
p60-S6K1 transcript and subsequent sequenc-
ing of the detected fragment following primers
were used: forward – TTCTGTCGGGAGTAG-
CACTG (encompasses the p60-S6K1 tran-
script specific insertion), reverse – GTGCTGT-
GGATTGGTGGAGT (against the exon 9 of
the primary S6K1 transcript) with the resulted
PCR product size of 787 bp. For the evaluation
of p60-S6K1, total S6K1 and β-actin mRNA
expression in a subset of cell lines by RT-PCR
following primers were used: 1) p60-S6K1:
forward – TTCTGTCGGGAGTAGCACTG
(the same oligonucleotide as for sequencing),
reverse – GTGCTGTGGATTGGTGGAGT
(against the exon 3 of the primary S6K1 tran-
script) with the resulted PCR product size of
257 bp. 2) Total S6K1 (exon 2-4): forward –
GACCATATGAACTTGGCATGG (against the
exon 2 of the primary S6K1 transcript), re-
verse – TCCCAGTATTTGCTCCTGTTAC
(against the exon 4 of the primary S6K1 tran-
script) with the resulted PCR product size of
174 bp. 3) Total S6K1 (exon 14-15): forward –
CACCTCGAAGATTTATTGGCA (against the
exon 14 of the primary S6K1 transcript), re-
verse – GTGCTCTGGCCGTTTGG (against
the exon 15 of the primary S6K1 transcript)
101
Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform
with the resulted PCR product size of 260 bp.
4) Total S6K1 (full-length): forward –
AGACAGGGAAGCTGAGGACA (against
the exon 1 of the primary S6K1 transcript),
reverse – GTGCTCTGGCCGTTTGG (against
the exon 15 of the primary S6K1 transcript)
with the resulted PCR product size of 1510 bp.
5) β-actin: forward – GGACTTCGAGCAAG-
AGAT, reverse – AGCACTGTGTTGGCGTAC
with the resulted PCR product size of 234 bp.
RT-PCR. Total RNA was extracted and pu-
rified from cell lines using GeneJET RNA
Purification Kit (#K0503, Thermo Scientific).
After extraction 1 µg of total RNA was treated
with DNase I (#EN0525, Thermo Scientific)
and further used to synthesize the first cDNA
strand with High-Capacity cDNA Reverse
Transcription Kit (#4374966, Applied
Biosystems). All procedures were performed
according to manufacturer’s instructions. PCR
was performed on 1/15 (1.5 µl) of cDNA in
25 µl of total reaction volume containing
0.2 mM dNTP mix, 10X DreamTaq Buffer
(#EP0701, Thermo Scientific), 0.5 µM of each
primer and 0.65 units of DreamTaq DNA
Polymerase (#EP0701, Thermo Scientific). For
PCR amplification of the full-length p70/p85-
S6K1 transcript following PCR mixtures were
prepared in total volume of 25 µl: 0.2 mM
dNTP mix, 5X Phusion HF Buffer (#F-530S,
Thermo Scientific), 3 % DMSO, 0.5 µM of
each primer and Phusion High-Fidelity DNA
Polymerase (#F-530S, Thermo Scientific).
PCR conditions were (i) 95°C for 2 min as
initial denaturation followed by 35 cycles of
denaturing at 95°C for 25 sec, annealing at
either 55°C (for β-actin) or 57°C (for S6K1
(exon 2-4, exon 14-15) and p60-S6K1) for
25 sec, extension at 72°C for 1 min and a final
extension at 72°C for 5 min for PCR amplifi-
cation with DreamTaq DNA polymerase; (ii)
98°C for 1 min, then 35 cycles of 98°C for
10 sec, 62°C for 25 sec (for full-length p70/
p85-S6K1) and 72°C for 1 min, followed by
5 min at 72°C for PCR amplification with
Phusion DNA polymerase. Resulting PCR
products were resolved on 1-2 % agarose gel.
Immunoblotting. Whole-cell lysates were
prepared by suspending cells in lysis buffer
(25 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM EDTA, 0.5 % Triton X-100, 5 % glyc-
erol, 1 mM Na3VO4, 2.5 mM Na pyrophos-
phate, 1 mM β-glycerophosphate supplemented
with a cOmplete EDTA-free protease inhibitor
cocktail tablet (Roche) and phosphatase in-
hibitors (Sigma-Aldrich)) with subsequent cen-
trifugation at 12,000 g for 10 min to obtain
lysates cleared of insoluble material. The pro-
tein content of cleared lysates was measured
with the Coomassie Protein Assay Reagent
(#1856209, Thermo Scientific). Samples were
resolved by 10 % SDS-PAGE and transferred
to a polyvinylidene difluoride (PVDF) mem-
brane (Immobilon®-P, Millipore). The mem-
brane was further incubated with primary anti-
bodies of appropriate dilutions. The antibody
against the S6K1 C-terminus was diluted 1:2500
[24] and dilution for the β-actin antibody was
1:10000 (clone 13E5, Sigma-Aldrich). Bound
antibodies were detected using horseradish
peroxidase-conjugated anti-rabbit or anti-mouse
antibodies followed by the enhanced chemilu-
minescence reagent (GE Healthcare). All the
secondary antibodies were diluted 1:10000.
Results and Discussion
Our recent study demonstrated that the p60
isoform of S6K1 can be alternatively trans-
102
I. V. Zaiets, V. V. Holiar, V. V. Filonenko
lated presumably from the common with p85/
p70 S6K1 transcript via usage of the third
in-frame start codon (Fig.1) [23]. Despite the
described source of p60-S6K1 translation, the
given isoform can be supposedly generated
from the alternative S6K1 mRNA splice vari-
ant termed as the S6K1 transcript variant 4
(further p60-S6K1 transcript) according to
the data of the NCBI Reference Sequence
Database (NM_001272044.1, RefSeq, NCBI).
A nucleotide sequence for this transcript was
derived from the data of NEDO human cDNA
sequencing project focused on splicing vari-
ants (AK297147.1, GenBank, NCBI) [unpub-
lished data]. This S6K1 transcript contains an
insertion between exons 1 and 2 (we called
it exon 1a) that bears an in-frame stop codon
(Fig. 1). The latter does not allow p70-S6K1
and p85-S6K1 to be translated causing pre-
mature termination of translation. However,
the full-length p60-S6K1 isoform can be
translated from this transcript (Fig. 1) by
utilizing the third in-frame translation start
codon located in exon 2. Nevertheless, iden-
tity of this transcript, termed p60-S6K1, has
not been elucidated, as well as its expression
in cells.
To identify the p60-S6K1 mRNA we em-
ployed a method of RT-PCR using total RNA
from MCF-7 cells as a source of the p60-S6K1
mRNA. For PCR detection one of the primers
targeted the p60-S6K1 mRNA-specific exon
1a, whereas another primer was complemen-
tary to a region of the exon 9. RT-PCR analy-
sis of MCF-7 total RNA revealed expression
of the p60-S6K1 transcript (Fig. 2A). The re-
sulted PCR product was extracted from a gel
and subjected to DNA sequencing. The results
of sequencing showed that the DNA fragment
amplified during PCR corresponded to a nu-
cleotide sequence of the S6K1 transcript vari-
ant 4 found in the NCBI Reference Sequence
Database (Fig. 2B).
After the identity of p60-S6K1 mRNA was
established, we investigated expression of this
transcript by RT-PCR in a number of cell lines
Fig. 1. Schematic demonstration of the p60-S6K1 splice variant relative to the pre-
dominant p60/p70/p85-S6K1 transcript. The transcript variant encoding the only p60-
S6K1 isoform contains an additional exon (designated as 1a) compared to the p60/p70/
p85-S6K1 transcript encoding all three isoforms that are translated from corresponding
alternative start codons. The 1a exon includes an in-frame stop codon that terminates
translation of p70/p85-S6K1 isoforms, thus forcing p60-S6K1 transcript to initiate
translation only the p60-S6K1 isoform from the third, nearest in-frame start codon.
103
Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform
of different origin. Additionally, we analyzed
expression of the p60-S6K1 isoform in these
cells in comparison with the protein and
mRNA expression levels. The data demon-
strated heterogeneous expression of the p60-
S6K1 mRNA in different types of cells
(Fig. 3A). Moreover, no correlation between
the expression levels of the p60-S6K1 mRNA
and total S6K1 mRNA was observed. This
implies that the p60-S6K1 isoform could play
a different role compared to the full-length
Fig. 3. Expression profile of the p60-S6K1 isoform in a
panel of cell lines. A) RT-PCR analysis of p60-S6K1
mRNA expression in different cell lines (MCF-7, HEK-
293, HeLa, HepG2, U-87, U-373, U-937, Jurkat). A sub-
set of the cell lines was subjected to a total mRNA isola-
tion and subsequent cDNA synthesis. Total cDNA for
each cell line was analyzed by PCR using primers spe-
cific to p60-S6K1 (top panel), total S6K1 (medium pan-
els) and β-actin (bottom panel). The resulted PCR prod-
ucts were separated by 2 % agarose gel electrophoresis.
B) Western blot analysis of p60-S6K1 protein expression
in the indicated cell lines. Whole-cell lysates were pre-
pared from the cell lines and loaded onto 10 % SDS-
PAGE (20 µg of each lysate except for 2 µg of MCF-7
lysate). Separated S6K1 was blotted against polyclonal
C-terminal S6K1 antibodies. β-actin was used to confirm
equal loading of protein in each lane.
A
B
A
B
Fig. 2. The RPS6KB1 gene gives rise to the transcript
variant (p60-S6K1 mRNA) supposedly encoding the
p60-S6K1 isoform. A) PCR detection of p60-S6K1
mRNA from MCF-7 cells. For PCR analysis a forward
primer was designed to be specific to a region of the in-
serted exon 1a, whereas a reverse one was complemen-
tary to a region of the exon 9. B) A sequenced PCR frag-
ment extracted from the gel represented in A) displays
inclusion of the exon 1a (sequence in blue). Primers used
for DNA sequencing (sequences in green) were the same
as for PCR analysis giving the PCR product of 787 bp in
length.
104
I. V. Zaiets, V. V. Holiar, V. V. Filonenko
p70/p85-S6K1 isoforms. To our surprise, how-
ever, usage of different primer pairs for detec-
tion of total S6K1 mRNA gave different ex-
pression profiles. Only a pair of primers pro-
ducing full-length p60/p70/p85-S6K1 mRNA
detected the expression levels which roughly
conformed to the protein expression levels
shown by immunoblotting, although the effi-
ciency of PCR employing these primers was
quite low. However, a question why the use of
different S6K1 primers provides varied the
results of S6K1 expression remains to be re-
solved in future studies since it is very impor-
tant for further quantitative analysis of p60-
S6K1 expression in cells and tissues.
The results also showed that p60-S6K1
mRNA expression levels do not correlate with
those of the p60-S6K1 protein (Fig. 3). The
absence of correlation between p60-S6K1
mRNA and protein expression may be account-
ed for a mechanism of p60-S6K1 generation,
since this isoform can also be initiated from the
common p60/p70/p85-S6K1 transcript.
Therefore, the ability of a cell to produce the
p60-S6K1 isoform using alternative pathways
might be responsible for such a difference be-
tween the p60-S6K1 mRNA and protein levels.
Unfortunately, we could not estimate the con-
tribution of the p60-S6K1 transcript expression
in the protein level of the p60-S6K1 isoform
applying the siRNA technology, since the p60-
S6K1-specific insertion represented a poor tar-
get sequence for RNAi.
In conclusion, we provided a new evidence
for the existence of the novel S6K1 transcript,
referred to as the p60-S6K1 transcript suggest-
ing existence of at least two alternative modes
in the p60S6K1 expression. The identified
transcript displays a differential pattern of
expression in different cell types. Yet, the ex-
pression of the p60-S6K1 mRNA does not
correlate with the total S6K1 mRNA indicating
that the former could play a distinct and inde-
pendent role in cellular physiology and S6K1-
related diseases.
This work was funded by the State Budget
Program «Support for the Development of
Priority Areas of Scientific Research» (Code:
6541230).
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106
I. V. Zaiets, V. V. Holiar, V. V. Filonenko
Ідентифікація нового сплайсового варіанту
S6K1, який кодує ізоформу p60-S6K1.
І. В. Заєць, В. В. Голяр, В. В. Філоненко
Мета. З’ясувати можливість існування сплайсової
мРНК кінази S6К1 специфічної виключно для р60-
S6К1 ізоформи. Методи. ЗТ-ПЛР, ДНК секвенування,
Вестерн-блоттинг. Результати. ЗТ-ПЛР аналіз тоталь-
ної РНК, виділеної із клітин лінії MCF-7, з наступним
секвенуванням ДНК виявив новий сплайсовий варі-
ант мРНК S6K1, що містить додатковий екзон 1а і
кодує лише p60 ізоформу S6K1. Для оцінки рівня
експресії p60-S6K1 мРНК та білку було застосовано
ЗТ-ПЛР та вестерн-блот. Результати вказують на
гетерогенну експресію транскрипту p60-S6K1, що не
корелює ні з експресією загальної S6K1 мРНК, ні з
білковим вмістом p60-S6K1 у досліджуваних клітин-
них лініях. Висновки. Надано докази існування нової
сплайсової мРНК S6K1, яка кодує ізоформу p60-S6K1,
що свідчить про існування в клітині двох незалежних
шляхів експресії p60-S6K1, а саме альтернативну
трансляцію спільної для головних S6K1 ізоформ
мРНК та/чи трансляцію нової специфічної лише для
р60-S6K1 сплайсової мРНК. Оскільки рівень екс-
пресії p60-S6K1 транскрипту в різних клітинних лі-
ніях не корелює із експресією загальної мРНК S6K1,
цілком можливо, що p60-S6K1 ізоформа може віді-
гравати відмінну від інших S6K1 ізоформ роль у
клітинній фізіології, а також при розвитку патологій,
які пов’язані із S6K1.
К л юч ов і с л ов а: p60-S6K1 транскрипт, альтерна-
тивний сплайсинг, експресія генів.
Идентификация нового сплайсового варианта
S6K1, который кодирует изоформу p60-S6K1.
И. В. Заец, В. В. Голяр, В. В. Филоненко
Цель. Выяснить возможность существования сплайсин-
говой мРНК киназы S6К1 специфической исключитель-
но для р60-S6К1 изоформы. Методы. ОТ-ПЦР, ДНК
секвенирование, Вестерн-блоттинг. Результаты. ОТ-ПЦР
анализ тотальной РНК, выделенной из клеток линии
MCF-7, с последующим секвенированием ДНК выявил
новый сплайсовый вариант S6K1, содержит дополни-
тельній екзон 1а и кодирует только изоформу p60-S6K1.
Далее были использованы ОТ-ПЦР и вестерн-блоттинг
для оценки уровней p60-S6K1 мРНК и белка. Результаты
показали гетерогенную экспрессию транскрипта p60-
S6K1, экспрессия которого не коррелировала ни с тоталь-
ной S6K1 мРНК, ни с белковым содержанием p60-S6K1
в исследуемых клеточных линиях. Выводы. Пре достав-
лены доказательства существования нового сплайсового
варианта S6K1, который кодирует изоформу p60-S6K1,
что указывает на существование в клетке двух независи-
мых путей експрессии p60-S6K1 изоформы, а именно
альтернативную трансляцию общей для основных изо-
форм мРНК и/или трансляцию новой специфичной лишь
для p60-S6K1 сплайсовой мРНК. Поскольку уровень
экспрессии p60-S6K1 транскрипта в различных клеточ-
ных линиях не коррелируют с экспрессией тотальной
S6K1 мРНК, можна предположить, что p60-S6K1 изо-
форма может играть отличную то других S6K1 изоформ
роль в клеточной физиологии, а также при развитии
патологий, которые связаны с S6K1.
К л юч е в ы е с л ов а: p60-S6K1 транскрипт, альтер-
нативный сплайсинг, экспрессия генов.
Received 25.11.2018
|
| id | nasplib_isofts_kiev_ua-123456789-154396 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T15:52:08Z |
| publishDate | 2019 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Zaiets, I.V. Holiar, V.V. Filonenko, V.V. 2019-06-15T15:06:01Z 2019-06-15T15:06:01Z 2019 Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform / I.V. Zaiets, V.V. Holiar, V.V. Filonenko // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 99-106. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00099B https://nasplib.isofts.kiev.ua/handle/123456789/154396 577 Aim. To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone. Methods. RT-PCR, DNA sequencing, Western blotting. Results. RT-PCR analysis of total RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1 splice variant that possesses additional exon 1a and encodes the p60 isoform of S6K1 only. Moreover, RT-PCR and western blotting were applied to estimate the expression levels of the p60-S6K1 mRNA and protein. A heterogeneous expression of the p60-S6K1 transcript was observed; it did not correlate with the total S6K1 mRNA expression and with the protein content of p60-S6K1 in the studied cell lines. Conclusions. We provide the evidence for the existence of a novel S6K1 splice variant coding for the p60-S6K1 isoform. The cell can employ two distinct pathways of the p60-S6K1 expression based on alternative translation of mRNA common for the main S6K1 isoforms mRNA and/or translation of the novel p60-S6K1 specific mRNA. Since the levels of the p60-S6K1 transcript expression do not correlate with the expression of total S6K1 mRNA, it is plausible that the p60-S6K1 isoform plays a role distinct from that of other isoforms in cellular physiology, as well as in the development of S6K1-related pathologies. Мета. З'ясувати можливість існування сплайсової мРНК кінази S6К1 специфічної виключно для р60-S6К1 ізоформи. Методи. ЗТ-ПЛР, ДНК секвенування, Вестерн-блоттинг. Результати. ЗТ-ПЛР аналіз тотальної РНК, виділеної із клітин лінії MCF-7, з наступним секвенуванням ДНК виявив новий сплайсовий варіант мРНК S6K1, що містить додатковий екзон 1а і кодує лише p60 ізоформу S6K1. Для оцінки рівня експресії p60-S6K1 мРНК та білку було застосовано ЗТ-ПЛР та вестерн-блот. Результати. вказують на гетерогенну експресію транскрипту p60-S6K1, що не корелює ні з експресією загальної S6K1 мРНК, ні з білковим вмістом p60-S6K1 у досліджуваних клітинних лініях. Висновки. Надано докази існування нової сплайсової мРНК S6K1, яка кодує ізоформу p60-S6K1, що свідчить про існування в клітині двох незалежних шляхів експресії p60-S6K1, а саме альтернативну трансляцію спільної для головних S6K1 ізоформ мРНК та/чи трансляцію нової специфічної лише для р60-S6K1 сплайсової мРНК. Оскільки рівень експресії p60-S6K1 транскрипту в різних клітинних лініях не корелює із експресією загальної мРНК S6K1, цілком можливо, що p60-S6K1 ізоформа може відігравати відмінну від інших S6K1 ізоформ роль у клітинній фізіології, а також при розвитку патологій, які пов’язані із S6K1. Цель. Выяснить возможность существования сплайсинговой мРНК киназы S6К1 специфической исключительно для р60-S6К1 изоформы. Методы. ОТ-ПЦР, ДНК секвенирование, Вестерн-блоттинг. Результаты. ОТ-ПЦР анализ тотальной РНК, выделенной из клеток линии MCF-7, с последующим секвенированием ДНК выявил новый сплайсовый вариант S6K1, содержит дополнительный экзон 1а и кодирует только изоформу p60-S6K1. Далее были использованы ОТ-ПЦР и вестерн-блоттинг для оценки уровней p60-S6K1 мРНК и белка. Результаты показали гетерогенную экспрессию транскрипта p60-S6K1, экспрессия которого не коррелировала ни с тотальной S6K1 мРНК, ни с белковым содержанием p60-S6K1 в исследуемых клеточных линиях. Выводы. Предоставлены доказательства существования нового сплайсового варианта S6K1, который кодирует изоформу p60-S6K1, что указывает на существование в клетке двух независимых путей экспрессии p60-S6K1 изоформы, а именно альтернативную трансляцию общей для основных изоформ мРНК и/или трансляцию новой специфичной лишь для p60-S6K1 сплайсовой мРНК. Поскольку уровень экспрессии p60-S6K1 транскрипта в различных клеточных линиях не коррелируют с экспрессией тотальной S6K1 мРНК, можно предположить, что p60-S6K1 изоформа может играть отличную то других S6K1 изоформ роль в клеточной физиологии, а также при развитии патологий, которые связаны с S6K1. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform Ідентифікація нового сплайсового варіанту S6K1, який кодує ізоформу p60-S6K1. Идентификация нового сплайсового варианта S6K1, который кодирует изоформу p60-S6K1. Article published earlier |
| spellingShingle | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform Zaiets, I.V. Holiar, V.V. Filonenko, V.V. Structure and Function of Biopolymers |
| title | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform |
| title_alt | Ідентифікація нового сплайсового варіанту S6K1, який кодує ізоформу p60-S6K1. Идентификация нового сплайсового варианта S6K1, который кодирует изоформу p60-S6K1. |
| title_full | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform |
| title_fullStr | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform |
| title_full_unstemmed | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform |
| title_short | Identification of a novel S6K1 splice variant coding for the p60-S6K1 isoform |
| title_sort | identification of a novel s6k1 splice variant coding for the p60-s6k1 isoform |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154396 |
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