Steady-state levels and degradation of cyclin mRNAs and some other cell cycle related mRNAs were measured at early G1, late G1, S and G2⁄ M phases.. Changes in mRNA stability accounted f
Trang 1to the changes in steady-state levels of cyclin mRNA in the mammalian cell cycle
Anna Penelova1, Larry Richman1, Barbara Neupert1, Viesturs Simanis2and Lukas C Ku¨hn1
1 Genetics Unit, Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland
2 Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland
Introduction
Cyclin-dependent kinases (cdks) are central to the
pro-gression and control of the mammalian cell cycle [1–3]
Their activity is regulated positively by interaction with
cyclins and negatively by cdk-inhibitors that bind to
cdk-cyclin complexes Cyclin-dependent kinases are
also regulated by phosphorylation The protein levels
of cdk activators and inhibitors are tightly controlled
by the rate of their synthesis and by specific
phos-phorylation events that initiate ubiquitination and
degradation by proteasomes, thus limiting expression
to a specific cell cycle phase D-type cyclins (D1, D2
and D3) are highest in early G1phase, when they
acti-vate cdk4 and cdk6 E-type cyclins (E1 and E2) peak
in late G1and associate with cdk2 to complete G1and initiate S phase Cyclin A2 accumulates during S phase with highest levels in late S and G2 It associates with cdk2 during S phase and subsequently with cdk1 (cdc2) to pass the S⁄ G2boundary Finally, progression through G2and mitosis require cyclins B1 and B2 that associate with cdk1
Because the expression of cyclins plays a large part
in controlling cell cycle progression, it is important to understand the transcriptional and post-transcriptional mechanisms that influence cyclin levels Indeed, recent microarray data demonstrate significant variations of cyclin mRNA levels in human fibroblasts after release from serum starvation (G0 phase) [4] or a double thymidine block (late G1 phase) [5] Transcription of
Keywords
cell cycle; cyclin; elutriation; fluorescence
activated cell sorter; mRNA stability
Correspondence
L C Ku¨hn, Swiss Institute for Experimental
Cancer Research, Genetics Unit, Chemin
des Boveresses 155, CH-1066 Epalinges,
Switzerland
Fax: +4121 652 69 33
Tel: +4121 692 58 36
E-mail: lukas.kuehn@isrec.ch
(Received 29 June 2005, accepted 16
August 2005)
doi:10.1111/j.1742-4658.2005.04918.x
Cyclins are the essential regulatory subunits of cyclin-dependent protein kinases They accumulate and disappear periodically at specific phases of the cell cycle Here we investigated whether variations in cyclin mRNA levels in exponentially growing cells can be attributed to changes in mRNA stability Mouse EL4 lymphoma cells and 3T3 fibroblasts were synchron-ized by elutriation or cell sorting Steady-state levels and degradation of cyclin mRNAs and some other cell cycle related mRNAs were measured at early G1, late G1, S and G2⁄ M phases In both cell lines mRNAs of cyclins
C, D1 and D3 remained unchanged throughout the cell cycle In contrast, cyclin A2 and B1 mRNAs accumulated 3.1- and 5.7-fold between early G1 and G2⁄ M phase, whereas cyclin E1 mRNA decreased 1.7-fold Mouse cyclin A2 and B1 genes, by alternative polyadenylation, gave rise to more than one transcript In both cases, the longer transcripts were the minor species but accumulated more strongly in G2⁄ M phase All mRNAs were rather stable with half-lives of 1.5–2 h for cyclin E1 mRNA and 3–4 h for the others Changes in mRNA stability accounted for the accumulation in
G2⁄ M phase of the short cyclin A2 and B1 mRNAs, but contributed only partially to changes in levels of the other mRNAs
Abbreviations
cdk, cyclin dependent kinase; DMEM, Dulbecco’s modified Eagle medium; DRB, 5,6-dichloro-1-b- D -ribofuranosylbenzimidazole; FACS, fluorescence activated cell sorter; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region.
Trang 2D-type cyclin mRNA is certainly induced by mitogenic
signals that trigger G0⁄ G1 transition [6], whereas
tran-scription of cyclin E1 starts in late G1 [7] Likewise,
A- and B-type cyclin mRNA were reported to be
induced in S and G2⁄ M phase as a consequence of
events in G1phase [4,8–10] In addition, several studies
concluded that cyclin, cdk and cdk-inhibitor mRNA
stability can vary throughout the cell cycle [11–15]
Cer-tain transacting proteins such as HuR were proposed
as regulators of changes in mRNA stability during the
cell cycle [15] In this context it is of interest that during
vertebrate evolution many of the cyclin mRNAs show
a rather high phylogenetic conservation of their 3¢
un-translated regions (3¢UTR) suggesting that specific
ele-ments in the 3¢UTR might contribute to control RNA
half-life [16] On the other hand a recent study with
human MOLT-4 cells showed no change in cyclin
mRNA half-lives throughout the cell cycle [17]
While most studies on cyclin mRNA stability in the
cell cycle have been carried out with human cells,
essential regulatory steps are likely to be conserved in
evolution and thus amenable to genetic analysis in the
mouse We therefore examined mRNA expression and
stability in synchronized mouse lymphoma EL4 cells
and 3T3 fibroblasts We analyzed the mRNA
steady-state level and half-life of mouse cyclins and a selection
of other cell cycle related genes for which important
cell cycle-related changes were reported in microarray
studies [4,5] We show that mRNAs for cks2, cyclin
A2, B1 and E1 vary in the cell cycle but that mRNA
half-life changes contribute only partially to these
vari-ations
Results
Steady-state levels of cyclin mRNA in the cell
cycle
In a first series of experiments we determined whether
mRNA steady-state levels of cyclins and several cell
cycle-related mRNAs change at different positions in
the cell cycle To achieve this, about 5· 108
logarith-mically dividing mouse EL4 lymphoma cells were
sep-arated by elutriation into 12–15 fractions EL4 cells
are particularly well suited for this separation method
as they are not adherent and grow to high density An
aliquot of each fraction was analysed on a fluorescence
activated cell sorter (FACS) for the profile of DNA
content after propidium iodide staining Pooled
frac-tions of cells highly enriched in early G1, late G1, S
and G2⁄ M phase were selected for further analysis
(Fig 1A) Steady-state mRNA levels were analysed by
real-time PCR By taking the early G1 cells as a
refer-ence, mRNA levels of cyclins C, D1 and D3, as well
as c-myc, RanGTPase and RanBP1 were unchanged (Fig 1B) Cyclin D2 was not expressed in EL4 cells Cyclin E1 mRNA increased slightly in late G1 and then diminished about 2-fold in G2⁄ M phase The clearest induction in G2⁄ M compared to early G1 cells was observed for cyclin A2 mRNA (3.1-fold) and cyclin B1 mRNA (5.7-fold) Cks2 mRNA was three-fold higher in S phase and 2.4-three-fold higher in G2⁄ M and very similarly the control histone H4 mRNA showed a threefold increase in S phase Thus, changes
in mRNA occur parallel to changes in protein expres-sion [18,19], but cannot account for strong differences
of cyclin protein levels that are modulated post-trans-lationally [20,21] Overall we observed smaller differ-ences in RNA steady-state levels than those reported
by others for human cells [11,12,15]
The relatively small changes in mRNA levels made
us wonder whether there was any problem with the separation procedure To verify this, we separated EL4 cells in logarithmic growth by the FACS according to cellular DNA content revealed by Hoechst 33342 (Fig 1C) This method gave highly enriched cell popu-lations with sufficient amount of mRNA for real-time PCR measurements, but could not distinguish early and late G1 cells The results were qualitatively very similar to the measurements obtained with elutriated cells, although somewhat less pronounced because we took the average G1 cells as a reference We found again that mRNA levels for cyclins C, D1 and D3 as well as for c-myc, RanGTPase and RanBP1 showed
no changes in the cell cycle (Fig 1D) Cyclin E1 mRNA decreased from G1 to G2⁄ M by a factor of 1.7-fold, whereas the mRNA of cyclin A2, cyclin B1 and cks2 increased 1.9-, 3.2- and 2.4-fold, respectively
We found similar results with mouse 3T3 cells that were either sorted by the FACS or synchronized by a double thymidine block They showed no change in steady-state levels for most mRNAs, with the excep-tion of a two- to 2.5-fold increase between G1 and
G2⁄ M for mRNAs of cks2, cyclins A2 and B1 (data not shown)
Next we wanted to be sure that cells were fully viable after elutriation To test this, elutriated cell frac-tions were brought back into cell culture for 2–8 h, at which time their DNA content was analysed by the FACScan (Fig 2) EL4 cells advanced synchronously
in the cell cycle without significant delay (Fig 2) The first fractions of cells harvested in the elutriation pro-tocol behaved like early G1 cells They enter S phase only after about 4 h of culturing, whereas later frac-tions comprise G1 cells that resumed S phase almost immediately and that we considered therefore as late
Trang 3G1 cells The FACS profiles allowed us to estimate the
total cycle to about 13 h of which about 6 h
corres-pond to G1 phase, about 3.5 h to S phase and another
3.5 h to G2 phase and mitosis This correlated well
with the estimated doubling time of EL4 cells in
log-arithmic growth
No major variation in the mRNA half-life of cyclin
mRNAs in the cell cycle
In order to test whether changes in mRNA
steady-state levels correlate with any changes in mRNA
sta-bility, we carried out half-life measurements on the
different elutriated cell fractions Transcription was
inhibited with
5,6-dichloro-1-b-d-ribofuranosylbenzimi-dazole (DRB) and mRNA measured at 0, 30, 60, 120
and 180 min by real-time PCR (Fig 3) Half-life
measurements showed no strong differences in mRNA degradation rates in the different cell cycle phases Only mRNA of cyclins A2 and B1 showed at most a 1.6-fold higher stability in S and G2⁄ M phases As a positive control, we found as expected a rapid degra-dation with a half-life of less than 1 h for the unstable c-myc mRNA, indicating that the transcription block
by DRB was effective Similar data were also obtained with actinomycin D or with EL4 cells enriched in spe-cific cell cycle phases by the FACS (data not shown)
Northern blot analysis of mRNA from fractions
of elutriated EL4 cells
We needed to confirm the real-time PCR data by nor-thern blots of RNA from elutriated cells The cell cycle distribution of the cell fractions is shown in Fig 4
Fig 1 Steady-state levels of cyclin mRNAs in enriched cell cycle fractions of mouse EL4 cells Cells were separated either by elutriation or
by cell sorting into G 1 , S and G 2 ⁄ M phase fractions (A) Cells were separated by elutriation into about 15 fractions The DNA content was measured after Hoechst staining by the FACS Representative fractions showed a strong enrichment for cells in early G1(a), late G1(b),
S (c) or G 2 ⁄ M phase (d) (B) The mRNA content of these fractions was quantified by real-time PCR and normalized to mARP0 mRNA Values
in early G 1 cells were set as 1 Results are the average of at least four experiments ± SD (C) Typical FACS profile of the DNA content of logarithmically growing EL4 cells stained by Hoechst 33342 Cells were separated by FACS sorting into three fractions as indicated (D) In each fraction, mRNAs were quantified by real-time PCR Values are normalized to mARP0 mRNA The amount of each mRNA in G 1 phase cells is set as 1 Results are the average of two experiments.
Trang 4Northern blot hybridizations were carried out for
genes that had shown differences of steady-state levels
in the cell cycle (cyclin A2, B1 and E1) The invariant
mRNAs of cyclin D3 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were analysed as controls
(Fig 4A) These blots revealed that there is more than
one transcript from mouse cyclin A2 and B1 genes In
the case of mouse cyclin A2, besides the more
abun-dant mRNA of 1.8 kb, there is a minor species of
3.0 kb For mouse cyclin B1 we see in addition to the
most abundant 1.7 kb mRNA, a 2.5 kb mRNA and a
very minor 2.1 kb mRNA Based on EST database
searches taking into account all 5¢ and 3¢ ends of
iden-tified cDNAs, we concluded that these mRNA
hetero-geneities arise from alternative polyadenylation This
was confirmed by control hybridizations with 3¢UTR
probes downstream of the first polyadenylation site
that consistently revealed only the longer transcripts
(Fig 5) Longer transcripts of cyclins A2 and B1 were also visible in mouse 3T3 cells and in mouse thymus and spleen, but were less clearly detectable in tissues with fewer proliferating cells (data not shown) In tes-tes two cyclin A2 transcripts and only the shorter cyclin B1 transcript were visible, in agreement with previous reports [22,23] The hybridizations with a coding region probe, after normalization to GAPDH expression, showed that the 1.8 kb cyclin A2 mRNA accumulated about 1.8-fold in S and G2⁄ M phase com-pared to early G1 phase, while the 1.7 kb cyclin B1 mRNA increased at most threefold (Fig 4B) More strikingly, the longer mRNA variants of both cyclins accumulated much more than the short ones and reached 35–45% of the total amount in late S and
G2⁄ M cells The 2.5 kb mRNA of cyclin B1 showed reproducibly a strong, up to 10-fold increase, while the magnitude of the 3.0 kb cyclin A2 mRNA increase
Fig 2 Cell cultures of mouse EL4 cell fractions after elutriation Immediately after elutriation selected cell fractions enriched in a given cell cycle phase (as indicated) were put back into culture for 2, 4, 6 or 8 h The cell cycle progression was analysed by FACS profiles of the DNA content of propidium iodide stained aliquots of cells.
Trang 5showed some variation between experiments (Fig 4 and Fig 6A) The reason for this is unclear At the same time cyclin E1 declined 2.2-fold
Based on northern blot analysis, as already deter-mined by real-time PCR, changes in steady-state levels were not associated with strong modifications in the mRNA half-life in different cell cycle phases (Fig 6B) Half-lives were about 2 h for cyclin E1 mRNA and 3– 4.5 h for the other transcripts in most cell cycle phases These values are close to those obtained by real-time PCR Only in the case of the short cyclin A2 and B1 mRNAs were the half-lives significantly prolonged in
G2⁄ M phase This change fully accounts for the accu-mulation of these transcripts in G2⁄ M phase For the long cyclin A2 and B1 mRNAs we found also a minor stability change that cannot account for their strong accumulation in G2⁄ M phase
Given the clear accumulation of cyclin A2 and B1 mRNAs in G2⁄ M compared to early G1 and the reports on human cells that demonstrated a strong dif-ference in mRNA stability in these phases [11,12,15], it seemed important to verify carefully mRNA half-lives
at the transition between G2⁄ M and early G1 phase For this, EL4 cells were arrested in mitosis by noco-dazole and then released for 0, 30, 60, 90, 120 or
180 min At least 75% of the arrested cells completed mitosis and divided within 3 h (Fig 7A) mRNA half-lives were measured at each time-point (Fig 7B) The results indicated no significant changes in mRNA half-life for most transcripts except the long transcript of cyclin B1 which appeared to decay quite rapidly at the time of the release Notably with the nocodazole arres-ted cells we did not find the prolonged half-life seen before for cyclin A2 and B1 mRNAs in enriched
G2⁄ M fractions Based on these data it seems unlikely that changes in steady-state levels can be attributed to transient changes in half-life
Discussion
The purpose of the present study was to analyse the contribution of post-transcriptional mechanisms in the cell cycle regulation of cyclin mRNAs Previous studies
on HeLa cells [11,12] and colorectal carcinoma RKO cells [15] had found strong mRNA stability changes
We reasoned that such a feature, if it was physiologi-cally important, should be conserved between human and mouse We therefore analysed the steady-state lev-els and mRNA stability at different points in the cell cycle of mouse 3T3 and EL4 cell lines The general conclusion of our analysis is that, in contrast to these earlier studies, but in agreement with a recent publica-tion on human MOLT 4 cells [17], most cyclin
A
B
Fig 3 Half-life of cyclin mRNAs in EL4 cell fractions enriched by
elutriation Cell fractions were put back into cell culture for about
30 min and incubated for 0, 30, 60, 120 or 180 min with DRB prior
to the isolation of total mRNA Remaining mRNA was measured by
real-time PCR and normalized to mARP0 mRNA The short-lived
c-myc mRNA served as a control (A) The mRNA half-life was
calcu-lated from linear regression on semi-logarithmic plots Results are
the average of three to four experiments ± SD (B) Alternatively,
data of decay of mRNAs showing the strongest changes in
steady-state levels (Fig 1) were plotted on a semi-logarithmic scale and a
single regression line calculated The intercept of the regression
line at log10of 50% ¼ 1.699, corresponds to the half-life The lower
and upper 95% confidence limits were at 0.75 and 1.5 times the
half-life.
Trang 6mRNAs show relatively small changes in steady-state levels and their degradation rates do not vary more than twofold during the cell cycle
The moderate regulation cannot be attributed to a lack of cell synchronization or cell viability (Fig 2) Two independent methods, elutriation and cell sorting gave excellent separation between G1, S and G2⁄ M cells and indicate overall very similar changes in mRNA steady-state levels (Fig 1) We found similar results for mouse EL4 lymphoma cells and 3T3 cells derived from different tissue types, suggesting that EL4 cells in spite of being tumour-derived with an exceptionally rapid cell cycle show the same basic fea-tures in terms of cyclin mRNA regulation as immortal-ized 3T3 fibroblasts
Several transcripts did not vary throughout the cell cycle This was the case for mRNAs of cyclins C, D1 and D3 which had a constant half-life at all stages of the cell cycle of about 4 h, as well as c-myc, RanBP1 and RanGTPase mRNAs This is consistent with
A
B
Fig 4 Northern blot analysis of cyclin mRNAs in all fractions of a typical elutriation experiment (A) The content of G1, S and G2⁄ M cells (in percentage on top panel) was determined for 12 consecu-tive fractions by propidium iodide staining and FACS mRNA of cyc-lins A2, B1, E1 and D3 along with the control GAPDH mRNA were quantified by northern blot hybridization with probes of the coding regions Numbers on the right indicate sizes of transcripts (B) Cyclin mRNA expression in different fractions was normalized to GAPDH mRNA and is reported relative to the first cell fraction (arbi-trarily set as 1).
Fig 5 Alternative polyadenylation in mouse cyclin A2 and B1 mRNA mRNA of nonsynchronized EL4 cells was isolated at differ-ent times indicated after transcription inhibition by DRB and analysed by northern blot hybridization The blot shown was sequentially hybridized with probes of the cyclin A2 coding region, cyclin B1 coding region, cyclin A2 3¢UTR, cyclin B1 3¢UTR and finally the GAPDH coding region probe The experiment shown is representative for three experiments with similar results.
Trang 7previous studies [24,25] cks2 mRNA showed an
increase in late S and G2⁄ M phase confirming previous
observations [26,27], but no cell cycle-dependent
change in stability The cyclin E1 mRNA was
increased in the G1phase of the cell cycle, but this was
not accompanied by changes in mRNA half-life This
result is similar to the one reported by others who
released cells from serum starvation [12] or
synchron-ized them by harvesting freshly divided cells [17] We
saw a reproducible increase for mRNAs of cyclins A2
and B1 as cells moved from G1 to G2⁄ M phase (Figs 1
and 4) However, northern blot hybridizations revealed
a more complex situation than in human cells, with
alternative transcripts produced by differences in polyadenylation sites (Figs 4 and 5) The 1.8-kb tran-script of cyclin A2 that corresponds to 60–90% of the total cyclin A2 mRNA increased only about twofold, whereas the 3.0 kb cyclin A2 mRNA was 2.5- to 7-fold more expressed in G2⁄ M (Figs 4 and 6) This was accompanied by about a twofold increased half-life in
G2⁄ M phase for both mRNAs (Fig 6B) Similarly, the 1.7-kb cyclin B1 mRNA showed only a two- to three-fold accumulation in G2⁄ M, whereas the 2.5-kb cyclin B1 mRNA was increased about 10-fold in G2⁄ M (Figs 4 and 6) The two mRNAs decayed with a half-life that was similar and at most twofold prolonged in
A
B
C
Fig 6 Half-life measurements of cyclin mRNAs by northern blot hybridizations (A) Cell fractions of elutriated EL4 cells (see cell cycle distri-bution on top) were incubated in the presence of the transcription inhibitor DRB for various lengths of time Total mRNA was isolated and analysed by northern blot hybridization (B) The signal intensity was quantified by a phosphorimager and normalized to GAPDH mRNA The half-life of each mRNA in different cycle phases is reported Results are the average of two independent experiments (for cyclin A2 meas-ured each twice) ± SD (C) Alternatively, results of long and short cyclin A2 and B1 mRNAs were plotted on semi-logarithmic graphs and a single regression line calculated The intercept of the regression line at log 10 of 50% ¼ 1.699, corresponds to the half-life The lower and upper 95% confidence limits were at 0.75 and 1.5 times the half-life.
Trang 8G2⁄ M phase We conclude that the changes in mRNA
half-life may explain almost fully the accumulation of
the short A2 and B1 transcripts as well as partially the
3.0-kb A2 transcript In contrast, the greater increase
of the 2.5-kb cyclin B1 transcript cannot be explained
by half-life changes It is possible that the majority of
the regulation of these transcripts may reside in the
choice of polyadenylation site
The lack of changes in mRNA stability for mRNAs,
which have quite long half-lives, raises the question of
how their levels decrease at the end of M-phase We
have to assume a prolonged period of transcription
inhibition to explain the decay of an mRNA that
fluc-tuates 10-fold in the cycle At present it is unclear
what triggers the decrease in the level of the 2.5-kb
cyclin B1 mRNA at the M⁄ G1 transition Mitotic
arrest and release experiments showed no systematic
acceleration in mRNA degradation near mitosis or shortly after (Fig 7) We found similar results with re-cultured cells after elutriation (data not shown) The relatively modest twofold changes for the half-lives of mouse cyclin A2 and B1 mRNAs observed here contrast with the large changes observed in earlier analyses on human HeLa and colorectal carcinoma RKO cells [11,12,15] When HeLa cells were synchron-ized after release from a thymidine⁄ amphidicolin block, the half-life of cyclin B1 mRNA measured after actinomycin D addition increased from 1.2 h in early
G1to 12 h in G2⁄ M phase [11] With the same method the cyclin A mRNA half-life was reported to change from 1.6 h in early G1to 12 h in late G1 and 8 h in S and G2⁄ M phase [12] Similarly, cyclin A and B1 mRNA half-lives increased in RKO cells from about 2.5 h in G1to 18 h in S phase after release from serum
A
B
transition between M and G1phase Cells were blocked with nocodazole in mitosis and released for 0, 30, 60, 90, 120 and
180 min (A) The cell cycle distribution of each cell population was determined by flow cytometry (B) At different times of release cells were incubated with DRB and mRNA decay was determined by northern blot hybridization (C) The mRNA half-life is reported for the various mRNAs at different time-points of the nocodazole release The results are the average of three experi-ments ± SD.
Trang 9starvation [15] However, no full return to the initial
rapid mRNA degradation rates in G1 was observed
when cells were cultured for a full cell cycle [15] The
overall conclusion from our study of no major changes
in cyclin mRNA degradation throughout the cell cycle
is consistent with a recent report on cyclin mRNA
half-lives in human MOLT-4 cells [17] Introducing a
novel synchronization procedure of gently harvesting
freshly divided cells, which should be unperturbed in
their logarithmic growth, the authors reported that
cyclin A2 and B1 mRNA accumulated about 8-fold in
G2⁄ M compared to early G1 phase, whereas mRNA
half-lives fluctuated between 1.5 and 2.5 h [17] In this
study the small variations of stability did not account
for the relatively strong fluctuations of cyclin A2 and
B1 mRNA, while here we propose that 2-fold changes
in mRNA levels for the major, short cyclin A2 and B1
mRNAs can be accounted for by stability changes
The discrepancies between the different studies may
be due to differences in cell lines or the techniques
used to synchronize them It is noteworthy that both
EL4 and MOLT-4 cells are derived from T-cell
lymphomas, whereas HeLa and RKO cells are
epithe-lial carcinomas, and 3T3 cells are of fibroblast origin
As is has been proposed that cyclin mRNA
stabiliza-tion depends on specific mRNA-binding proteins
interacting with AU-rich regions, notably HuR [15], it
is conceivable that different cell lines express these
proteins differently Alternatively, HuR might be
induced only under certain conditions of stimulation
after a cell growth arrest, but not in the case of
separ-ation of cells during logarithmic growth Concerning
the techniques alone we found for EL4 cells, that
elutriation was less perturbing than other methods,
including release after double thymidine block or
nocodazole arrest where usually a large fraction of
cells (sometimes up to 30%) were unable to resume
growth It was reassuring that cell sorting gave results
similar to elutriation, as transcription inhibitor is
added to unperturbed logarithmically growing cells
and RNA isolated immediately after sorting
elimin-ating artefacts of cell culture
The similarity of our conclusions concerning mRNA
half-life to those reached by others with MOLT-4 cells
[17] seems to exclude fundamental differences in cyclin
mRNA decay between species However, there are
dif-ferences in the relative accumulation of cyclin A2 and
B1 mRNA, and the detection of multiple mRNA
species in the mouse was a surprise given previous
observations in human cells Others have reported
het-erogeneity of A2 and B1 transcripts in rodent species
[28,29] Human cyclin A mRNA shows mainly one
band [19] corresponding to the 2.7-kb mRNA in the
mouse, and human cyclin B1 mRNA is also a single species [18] corresponding to the 1.7-kb transcript in the mouse We show that mouse transcripts differ only
in their 3¢UTR, but not the coding regions Therefore, the significance of 3¢UTR differences is unclear They may perhaps play a role in localizing the mRNA or regulate its translation Future studies will address this
In conclusion, our study shows that post-transcrip-tional regulation contributes 2-fold to the short ver-sions of cyclin A2 and B1 mRNA, whereas minor fluctuations of mRNA levels in the other genes are possibly transcriptionally controlled The cyclic accu-mulation of the longer mouse cyclin A2 and B1 mRNAs may result from a combination of changes in alternative polyadenylation, transcription and minor mRNA stabilization in G2⁄ M phase
Experimental procedures
Cell culture Mouse 3T3 fibroblasts kindly provided by A Trumpp (ISREC, Switzerland) were grown in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v⁄ v) heat-inactivated fetal bovine serum (FBS; Sigma, St Louis, MO, USA) Mouse EL4 lym-phoma cells were grown in DMEM⁄ 5% (v ⁄ v) FBS Media were supplemented with 1% (w⁄ v) penicillin-streptomycin (Invitrogen) Cells were grown at 37C in a humidified atmosphere of 5% (v⁄ v) CO2
Cell synchronization protocols Cell sorting
Mouse 3T3 fibroblasts or EL4 lymphoma cells were grown
in logarithmic cell cultures for 2 days For mRNA steady-state measurements, cells were stained with 5 lgÆmL)1 Hoechst 33342 (Sigma, Franklin Lakes, NJ, USA) for
30 min, and separated into G1, S and G2⁄ M phase cells by FACS (FACStar Plus Pulse Processor; Becton-Dickinson, xxxx, xxxx) Alternatively, for mRNA half-life measure-ments, cells were incubated with 6 lgÆmL)1actinomycin D (Sigma) or 20 lgÆmL)1 DRB (Sigma) for 0, 30, 60 or
120 min, then stained with 5 lgÆmL)1 Hoechst 33342 for
30 min, and separated into G1, S and G2⁄ M phase cells by FACS Fractionated cells were collected in RLT buffer (Qi-agen, Valencia, CA, USA) and stored at)70 C until RNA extraction
Centrifugal elutriation Centrifugal elutriation was performed in a Beckman JE 5.0 centrifuge and a JE-5S rotor equipped with the standard
Trang 10separation chamber Logarithmically growing EL4 cells
(3· 108)6 · 108
) were introduced into the separation
chamber Cells were elutriated at 4C in NaCl ⁄ Pi
contain-ing 2% (v⁄ v) FBS Elutriation was executed at a constant
rotor speed of 2800 r.p.m (755 g) The fractionation of
cells into cell cycle subpopulations was accomplished by
increasing the pump speed stepwise from the initial flow
rate of 20 mLÆmin)1 to a maximum of 55 mLÆmin)1 Cell
fractions of 100 mL were harvested The cell cycle
distribu-tion was determined on propidium iodide-stained aliquots
of cells Enriched cell populations were cultured in fresh
DMEM⁄ 5% (v ⁄ v) FBS for 2, 4, 6 or 8 h and aliquots
re-analysed for the cell cycle distribution
Nocodazole block
Mouse EL4 cells (3· 105
) were grown in 75-cm2 flasks for
1 day Then they were incubated with 40 ngÆmL)1
nocodaz-ole (Sigma) for 14 h and released from the block for 30, 60,
90, 120 or 180 min To each fraction 20 lgÆmL)1 DRB
(Sigma) was added for 0, 30, 60, 120 and 180 min
One-tenth of each fraction was stained with propidium iodide
(Sigma) and analysed by FACS The rest of the cells were
stored at)70 C in RLT buffer (Qiagen) until RNA
extrac-tion
Propidium iodide staining
To test the cell cycle distribution of cells, an aliquot of cells
was fixed with ethanol, stained with 50 lgÆmL)1 propidium
iodide (Sigma) and 200 lgÆmL)1 RNase A (Sigma), and
analysed by a Becton-Dickinson FACScan flow cytometer
RNA extraction, RNA half-life measurements and
northern blot hybridization
Total cellular RNA was extracted with RNeasy Mini
Kit (Qiagen) following the manufacturer’s protocol For
RNA half-life measurements, 6 lgÆmL)1 actinomycin D or
20 lgÆmL)1 DRB was added 0, 30, 60, 120 and 180 min
before RNA extraction Total RNA (10 lgÆsample)1) was
separated on a 1.2% agarose formaldehyde gel RNA was
transferred by capillarity onto ImmobilonTM-Ny+
mem-brane (Millipore, Billerica, MA, USA) and UV-crosslinked
in a Stratalinker (Stratagene, La Jolla, CA, USA) at
1.2· 105lJ
The mouse cyclin A2, B1, D3, E1 and cks2 cDNA probes
were generated by PCR amplification of their coding
sequences The GAPDH probe template was the
EcoRI-HindIII fragment of the coding region [30] To detect long
transcripts of cyclins A2 and B1, we amplified by PCR the
3¢UTR of cyclin A2 at nucleotides 2129–2779 (GenBank
accession no NM_009828) and of cyclin B1 at nucleotides
2011–2311 (GenBank accession no NM_172301)
Fifty nanograms of DNA template was labelled by ran-dom priming for 4 h at 37C in a 30-lL reaction volume containing 50 mm Tris⁄ HCl pH 8, 200 mm Hepes, 0.1 mgÆmL)1 BSA, 5 mm MgCl2, 100 lm 2-mercaptoetha-nol, 20 lm dATP, 20 lm dGTP, 20 lm dTTP and 50 lCi [32P]dCTP (3000 CiÆmmol)1), 2 U Klenow (Roche, Basel, Switzerland) and 27 A260units hexanucleotide (Pharmacia, Peapack, NJ, USA) for priming
Membranes were prehybridized for at least 1 h at 42C
in hybridization buffer (50% formamide, 1% SDS, 4.8· NaCl ⁄ Cit, 10% dextran sulfate) with 100 lgÆmL)1 sal-mon sperm DNA Denatured probe was added to the hybridization solution and allowed to hybridize to the membrane at 42C overnight Membranes were rinsed with
1· NaCl ⁄ Cit, 0.1% SDS, washed twice for 30 min at
65C in 0.2 · NaCl ⁄ Cit, 0.1% SDS and then visualized by Imaging Plate BAS-MP 2040S (Fuji Photo Film, Tokyo, Japan) and Kodak BiomaxTMfilms (Rochester, NY, USA) Images were quantified using a Bio-imaging analyser
BAS-1000 (Fuji) and advanced image data analyzer (aida 2.0) software For additional hybridizations, the membranes were stripped twice for 15 min in 250 mL of boiling 0.1· NaCl ⁄ Cit, 0.5% SDS
Reverse transcription and real-time PCR
To avoid amplification of residual genomic DNA, firstly this was removed from total RNA on the RNeasy Mini Kit column by treating with RNase-free DNase I Set (Qiagen) according to the manufacturer’s protocol We then used a specific fluorogenic probe labelled with 5¢ 6-carboxy-fluor-secein (FAM) and 3¢ 6-carboxy-tetraethyl-rhodamine (TAMRA) for Taqman quantification Almost all fluoro-genic probes were chosen such as to hybridize to an exon– exon junction (Table 1), except in the case of cks2 mRNA where the reverse primer is at the intron–exon junction and histone 4 mRNA that has no intron To avoid cross-ampli-fication of pseudogenes, we verified that primer sequences did not appear elsewhere in the EST database When choosing a new primer set for real-time PCR we always verified that there was no significant amplification product
in the absence of reverse transcriptase
First-strand cDNA was synthesized using 1 lg RNA in a 20-lL reverse transcriptase reaction mixture, containing
1· RT buffer, 0.01 m dithiothreitol, 0.5 mm of each dNTP,
1 lg random hexamer pd(N)6 (Pharmacia), 2 U RNasin (Amersham Biosciences, Piscataway, NJ, USA) and 200 U M-MLV reverse transcriptase (Invitrogen) The reverse tran-scriptase reaction was carried out at 42C for 90 min and then inactivated for 5 min at 95C The cDNA was diluted
at least 20-fold prior to PCR amplification The PCR was performed in the GeneAmp5700 sequence detection sys-tem (Applied Biosyssys-tems, Foster City, CA, USA) Taqman PCR reactions were performed in a 25-lL volume