MOLECULAR AND CELLULAR BIOLOGY, May 1992, p.

2203-2212

Vol. 12, No. 5

0270-7306/92/052203-10$02.00/0

Copyright (C 1992, American Society for Microbiology

Selective Translational Control and Nonspecific Posttranscriptional

Regulation of Ribosomal Protein Gene Expression during
Development and Regeneration of Rat Liver
RONIT ALONI,t DAVID PELEG,4 AND ODED MEYUHAS*
Department of Developmental Biochemistry, Institute of Biochemistry, Hebrew
University-Hadassah Medical School, Jerusalem 91010, Israel
Received 14 November 1991/Accepted 13 February 1992

Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent
can be induced in the adult liver by partial hepatectomy.
In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and
regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about
70%o upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition,
the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines
during development through a posttranscriptional mechanism. When liver cells commence growth following
partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very
efficient recruitment into polysomes. The concomitant increase in the abundance of rp mRNAs under these
circumstances is achieved by a posttranscriptional mechanism. The apparent fluctuations in the translation
efficiency of rp mRNAs are accompanied by parallel changes in the expression of the genes encoding the
initiation factors eIF-4E and eIF-4A. Our results indicate that selective translational control of rp mRNAs in
mammals is not confined to manipulated cells in culture but constitutes an important regulatory mechanism
operating in vivo in the course of liver development and regeneration.
state in the adult. However, extensive proliferation

The biosynthesis of ribosomes in vertebrates is primarily

coordinated with the cellular growth status and requires an
equimolar accumulation of four rRNA and about 80 different
ribosomal protein (rp) molecules. This stoichiometry is
maintained by coordinate regulation at various levels of gene
expression from transcription to protein turnover (4, 31, 43,
49). Clearly, the translational control of rp mRNAs is the
most prevalent regulatory mechanism of vertebrate rp gene
expression and operates under a variety of physiological
conditions (reference 41 and references therein). Recently,
we have shown that the 5'-terminal pyrimidine tract, adjacent to the cap site of all vertebrate rp mRNAs rigorously
analyzed thus far, plays a critical role in their coordinate
translational control (41).
Posttranscriptional control of rp genes, most probably
through stability of the corresponding transcripts, has been
reported to occur in anucleate Xenopus embryos (62) and in
dexamethasone-treated rats (21). In addition, control at the
RNA processing level is evident for rpL1 in Xenopus laevis
(10). Finally, a balanced accumulation of ribosomal proteins
(r-proteins) is also achieved by modulating their turnover, as
observed in mouse oocytes (39) and differentiating rat myoblasts (30).
The development of rodent liver is associated with progressive decrease of mitotic activity to almost undetectable
levels in the adult tissue (reference 46 and references therein). Concomitantly, the hepatocytes acquire differentiated
functions and lose others. These developmental changes

involve extensive alterations in expression of numerous

liver-specific genes, as well as housekeeping genes, at the
transcriptional and posttranscriptional levels (61, 63).
Although the adult liver is normally quiescent, it retains
the ability to proliferate, as evident by its rapid regeneration
following removal of 70% of its mass. The regenerative
response in rodents consist of two stages: hypertrophia
lasting approximately 12 to 16 h, during which the expression of a large number of genes is initiated with a concomitant rise in the rate of RNA and protein synthesis; and a
subsequent hyperplasia characterized by a peak in DNA
synthesis at about 24 h and mitosis beginning 6 to 8 h later
(11, 22, 55). The transition from resting to growing state is
accompanied in the regenerating liver, as in other cells
undergoing similar transitions, with increased ribosome biosynthesis, which peaks at 12 to 18 h postoperation (14, 15,
73). The increase in synthesis rate of r-proteins in the
regenerating liver is associated with elevation of the abundance of the respective mRNAs (20, 56). This mode of
regulation differs from that observed in cultured mammalian
cells commencing growth, in which the synthesis of r-proteins is regulated solely at the translational level (35, 51,
53).
The experiments described below were designed to examine whether the relative cessation of liver cell proliferation,
upon development from fetal to adult life, and the resumption of liver growth during regeneration are accompanied by
modulation of rp gene expression and, if so, at what level.
Our results indicate that the expression of these genes is
selectively modulated at the translational level, in parallel to
changes in the abundance of the mRNAs encoding the
initiation factors eIF-4E and eIF-4A, and is posttranscriptionally regulated in a nonspecific manner.

*
Corresponding author.
t Present address: Department of Cell Biology, The Weizmann
Institute of Science, Rehovot 76100, Israel.

t Present

address:

Department

of Obstetrics and

Gynecology,

Central Emek Hospital Afula 18101, Israel.

2203

2204

ALONI ET AL.

MATERIALS AND METHODS

Animals. Pregnant female and adult (6- to 8-week-old)
male Sabra rats (Wistar origin) were obtained from the
Hebrew University breeding center. Fetal age was measured
from time of coitus which, because of variability in fertilization and implantation, is accurate to within 1 or 2 days.
Partial hepatectomy resulting in the removal of 70% of the
liver mass was performed on male rats as described by
Higgins and Anderson (29). Sham-operated rats were laprotomized, and their livers were manipulated but not excised.
Cell culture and labeling of RNA with [3H]uridine. Mouse
Ltk- cells were grown in monolayer as described previously
(21). For labeling the RNA, a confluent culture in 100-mm
dishes was incubated for 2 h at 37C with [5,6-3H]uridine
(37.5 Ci/mmol; Du Pont Co.) at 100 ,uCi/ml in 5.0 ml of
culture medium.
RNA extraction and analysis. Cytoplasmic RNAs from rat
liver and from Ltk- cells were extracted as described by
Schibler et al. (67) and Shaw et al. (68), respectively.
Isolation of the poly(A)+ mRNA and quantitative RNA
(Northern) blot analysis were performed as previously described (53). Fractionation of cytoplasmic RNA by sucrose
gradient centrifugation was carried out as described elsewhere (52).
Polysomal fractionation. Samples of about 100 to 200 mg
from fetal or adult rat liver were excised, quickly frozen in
liquid nitrogen, and kept at -70C. Samples were thawed in
5 volumes of polysomal buffer (0.14 M sucrose, 25 mM
Tris-HCl [pH 7.5], 10 mM MgCl2, 25 mM NaCl, 0.05%
Triton X-100, 100 ,ug of heparin per ml). Homogenization in
a Dounce homogenizer was carried out first with a loosefitting pestle (B) and, after addition of 1/9 volume of 10%
Triton X-100-10% deoxycholate to the homogenate, with a
tight-fitting pestle (A). Nuclei were pelleted by centrifugation for 2.5 min in a microfuge at 4C. The postnuclear
supernatant was diluted with an equal volume of polysomal
buffer containing 500 ,ug of heparin per ml. One milliliter of
this suspension was layered over a sucrose gradient, prepared as previously described (53). The gradients were
centrifuged at 27,000 rpm for 225 min at 4C in a Kontron
TST 28.38 swing-out rotor. After centrifugation, the gradients were collected from the bottom in 0.5-ml fractions into
tubes containing 5 ,u of 10% sodium dodecyl sulfate (SDS).
Extraction of the RNA from the polysomal and subpolysomal fractions was performed as described by Meyuhas and
Perry (52).
Nuclear runon transcription assay. Nuclei were prepared
from fetal and adult rats as described by Meisner et al. (47).
Elongation of nascent RNA chains in the isolated nuclei was
carried out essentially as described by Schibler et al. (66)
except for the absence of heparin sulfate and lower
(NH4)2SO4 concentration (35 mM) in the reaction mixture.
Labeled RNA was extracted from the reaction mixture (25)
and hybridized to GeneScreen Plus (New England Nuclear)bound DNA. Immobilization of plasmid DNAs to the filter
was carried out as previously described (21), using a slot blot
apparatus (Schleicher & Schuell). Plasmid-bearing filters
were prehybridized at 65C for overnight with 50 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES;
pH 7.5), 0.5 M NaCl, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 10 mM UTP, 0.2% SDS, 10 mM EDTA, and 300 ,ug of
salmon sperm DNA per ml. The 32P-labeled RNA (8 x 106 or
1 x 105 to 8 x 105 cpm for analysis of transcripts of
polymerase II or I, respectively) was added, and hybridization was conducted at 65C for 48 h. The blots were washed

MOL. CELL. BIOL.

with three changes of 5x SSC (lx SSC is 0.15 M NaCl plus

0.015 M sodium citrate)-0.2% SDS-0.5 mM EDTA for 5 min
each at room temperature and then twice for 15 min each
with the same solution at 65C and twice for 20 min each with
2x SSC at 65C. Blots were treated with 10 ,ug of RNase A
per ml in 2x SSC for 30 min at room temperature, washed
twice for 5 min each time with 2x SSC at room temperature,
and autoradiographed. The procedures used for isolation of
nuclei from regenerating and sham-operated livers, as well
as the runon transcription assay using these nuclei, were as
previously described (21). The autoradiographic signals were
quantified by densitometric scanning using a Helena Quickscan R & D densitometer (Helena Laboratories, Beaumont,
Tex.). Exposures were chosen so that the signals were
within the linear response range of the film.
Molecular probes. The isolated fragment probes used in
the Northern blot analysis were a 0.51-kb SacII-XbaI fragment containing a mouse rpL30 processed gene derived from
plcXba (78); a 0.97-kb fragment bearing the rpL32 processed
gene, 4A (17), joined to the 5' and 3' flanks of p3A; 0.29- and
0.33-kb EcoRI-HindIII fragments containing the cDNA inserts of mouse rpS16 and mouse rpL18a, respectively,
derived from subclones in pUC8 (2) of the original clones
(52); a 0.65-kb BamHI fragment containing human rpP2
cDNA (65); 0.37- and 0.73-kb PstI fragments spanning the
entire rat rpL5 cDNA (13); two 0.45-kb PstI fragments
containing rat rpL7 cDNA (42); a 1.15-kb PstI fragment
containing mouse cx-actin cDNA (54); a 0.62-kb PstI fragment containing human superoxide dismutase I (SOD)
cDNA (69); a 1.05-kb PstI fragment bearing rat 0-tubulin
cDNA (23); a 1.2-kb HindIlI fragment containing rat albumin
cDNA (36); a 1.6-kb PstI fragment containing phosphoenolpyruvate carboxykinase (PEPCK) cDNA (80); a 1.4-kb
EcoRI-HindIII fragment containing murine eIF-4E cDNA
(40); and 0.4-kb EcoRI fragment containing the 5' end of
mouse eIF-4A cDNA (57). Recombinant plasmids used in
the runon transcription assay contained the following inserts: rat rpL5 cDNA (13), rat rpL7 cDNA (42), rat rpL19
cDNA (12), rat rpL18a cDNA (5), mouse rpS16 and rpL32
cDNAs (52), human rpP2 cDNA (65), mouse a-actin cDNA
(54), rat PEPCK cDNA (80), pB5 containing a 1.15-kb
BamHI-EcoRI fragment from mouse 18S rDNA (9), and
plla.2 containing a 7.1-kb EcoRI fragment spanning the 3'
region of 18S, internal transcribed spacers, 5.8S, and most of
the 28S sequence from Chinese hamster rDNA (75).
RESULTS
The translation of rp mRNAs is repressed during liver
development and reactivated in regenerating liver. Selective
regulation of rp mRNA translation seems to be the prevalent
mechanism operative in most cases of transitions between
growing and nongrowing states of cultured mammalian cells
studied thus far (reference 41 and references therein). However, such a mode of regulation for rp mRNAs has not been
demonstrated to operate in whole animals. The mitotic
activity in the adult liver is negligible compared with that
observed in the fetal liver (24, 46). Therefore, we examined,
by monitoring the relative loading of rp mRNAs onto polysomes, whether the translation of rp mRNAs is affected
during liver development. Polysomes from fetal and adult
liver were size fractionated by sucrose gradient centrifugation. In both fetal and adult liver, approximately 77% of the
ribosomes are engaged in polysomes, as judged by the
proportion of total ribosomal particles, including subunits,
that sediments in the polysomal region of the gradient (Fig.

VOL. 12, 1992

RIBOSOMAL PROTEIN GENE EXPRESSION IN RAT LIVER

Fetal liver

Adult liver

2205

regenerating liver

E
cs

0
to

.0
:
0

.0

b
F

SOD

rpL5
-jm.f.

.,.fik:

-- .
A

I_._.;
II

. ...

**
--..,;

w~~~~~s

...

_1

U'

PH
1

11

g9

11

11

l9

Fraction number
FIG. 1. Repression of the translation of rp mRNAs during liver development and reactivation by partial hepatectomy. (a) Absorbance
profiles of polysomes and subpolysomal particles fractionated in sucrose gradients. A postnuclear supernatant from fetal (gestation day 18),
adult, and regenerating liver (15 h postoperation) was centrifuged through a 35-ml 15 to 45% sucrose gradient. The gradient was collected from
the bottom (left hand of the curve) in 0.5-ml fractions. The bottom 2 ml was discarded, and the A260 of each fraction was manually monitored
(squares). Approximately every six consecutive fractions were combined to yield 12 fractions of about 3 ml each (fractions 1 to 12). M,
monosomes; 60, 60S; 40, 40S. The absorbance profiles shown are representative of two to three profiles obtained for each treatment. Because
of variations in the amounts of liver cytoplasmic extract applied to each gradient, only qualitative comparisons between the absorbance
profiles can be made. (b) Distribution of rp and non-rp mRNAs along the gradients presented in panel a. RNAs isolated from fractions 1 to
12 were used for Northern blot analysis and hybridized with labeled cDNAs encoding rpL32, rpL5, and SOD. F, fetal liver; A, adult liver;
PH, partial hepatectomy. For each treatment, the same RNA preparations were hybridized with the different probes. The vertical dashed line
separates the polysomal fractions (left) and the subpolysomal fractions (right).
F

la). This constant proportion of polysome-associated ribosomes, observed in the liver, is in a sharp contrast to the
significant decrease in this proportion when measured in
growth-arrested mouse P1798 lymphosarcoma cells (53) and
NIH 3T3 fibroblasts (35). The polysomal association of
various mRNAs was assessed by Northern blot analysis of
each gradient fraction. Our results demonstrate a striking
difference in the polysomal distribution of the mRNAs
encoding rpL32 or rpL5 compared with that encoding SOD.
Thus, the proportion of rpL32 mRNA associated with polysomes decreases from 71% in the fetus to 17% in the adult,
and that of rpL5 mRNA from 76 to 30%. The SOD mRNA,
on the contrary, is loaded with a similar number of ribosomes, regardless of the developmental stage, exhibiting a
very efficient translation (about 95%) in polysomes in both
fetal and adult liver. Albumin mRNA, like that of SOD,
exhibits similar ribosomal loading in both developmental
stages (50). The results presented in Fig. lb and 2 indicate
that the repression of rp mRNA translation in the adult liver
involves mRNAs encoding basic r-proteins of both large (L5,
L32, and L7) and small (S16) subunits, as well as an acidic
protein (P2), but not mRNAs encoding other housekeeping
proteins (SOD, f-actin, and 1-tubulin) or a tissue-specific
protein (PEPCK). The reason for the relative augmented
ribosomal loading on rpL7 mRNA in the sham-operated
animal (Fig. 2) is not known. However, exceptional translational behavior of this mRNA has been previously reported
(53). It should be noted that the SOD mRNA (containing 153
codons [69]) is significantly smaller than those encoding
rpL5 and rpL7 (containing 296 [13] and 258 [42] codons,
respectively). However, unlike the rp mRNAs, it is fully

S16

SO

A
S

I P

I P

P2

Actin
Tubuiin
SOD

PEPCK

PH

ST P

A*

% in polysomes
F A SO PH
71 24 64

98

71 25 40

96

79 29 38 100
98

90 100100

98 84 85 100

100 91
85

97 100

100

100

FIG. 2. Polysome-subpolysome distribution of various mRNAs.

Cytoplasmic extracts from livers of 18-day fetal (F), adult (A),
sham-operated (SO), and 15-h partially hepatectomized (PH) rats
were centrifuged through sucrose gradients and separated into
polysomal (P) and subpolysomal (S) fractions. Poly(A)+ mRNA
from equivalent aliquots of these fractions was analyzed by Northern blot hybridization with the probes indicated at the left. Because
of variations in film exposure time for 3-actin mRNA, quantitative
comparisons of the autoradiographic signals can be made only
between the polysomal and subpolysomal fractions of the same
gradient. The relative amount of mRNA in the polysomal fraction of
the various gradients was determined by densitometric scanning of
the autoradiograms. Exposures were chosen so that the signals were
within the linear response range of the film. The results are presented at the right as the percentage of total mRNA in the polysomal
fraction.

2206

ALONI ET AL.

loaded with ribosomes even in the resting adult liver cells

(Fig. lb). Thus, the selective unloading of ribosomes from rp
mRNAs in adult liver cannot be simply ascribed to their
shorter coding sequences; father it reflects the distinctive
behavior of these two classes of mRNA.
In addition to the selective repression of the translation of
rp mRNAs during liver development, they are also distinguishable by their overall lower translation efficiency. This
feature is illustrated by their underrepresentation in polysomes, even in fetal liver (Fig. lb and 2). Hence, it appears
that in fetal liver, like in exponentially growing mammalian
cells in culture, about 25% of the rp mRNAs is translationally inactive (Fig. lb and 2) (35, 51, 53).
The overall amounts of RNA (polysomal plus subpolysomal) from fetal and adult liver used in the experiment
presented in Fig. 2 were similar. Hence, it seems that the
decrease in the translation efficiency of rp mRNAs during
development is accompanied by a substantial decline in their
abundance (a detailed analysis of this latter mode of regulation is presented later).
When the liver reaches the adult stage, it becomes quiescent yet retains the capacity to resume proliferation following partial hepatectomy. It has been previously shown that
the relative abundance of various rp mRNAs is increased
two- to threefold within 12 to 18 h postoperation (20, 56).
However, this elevation in the rp mRNA level cannot
account by itself for the total increase (about 10-fold) in the
synthesis rate of r-proteins (73). Thus, we set out to verify
whether partial hepatectomy also affects the translation
efficiency of rp mRNAs. The overall translation efficiency in
the regenerating liver, as indicated by the percentage of
ribosomes engaged in polysomes (78%), is similar to that
observed in liver of control adult (Fig. la) or sham-operated
(data not shown) animals. However, the ribosomes in the
regenerating liver are associated with heavier polysomes
(Fig. la). The results shown in Fig. lb indicate that the
mRNAs encoding rpL32 and rpL5 are efficiently (more than
90%) recruited into polysomes within 15 h postoperation. A
similar elevation in the translation efficiency has been observed for other rp mRNAs encoding L7, S16, and P2 (Fig.
2). The proportion (about 95%) of polysome-associated rp
mRNAs, observed during liver regeneration, is significantly
higher than that measured in the fetal liver (Fig. lb and 2) or
proliferating cells in culture (2, 35, 51, 53). Thus, partial
hepatectomy leads to increased translation efficiency of rp
mRNAs, which is comparable with that of mRNAs encoding
other housekeeping proteins such as SOD, 1-actin, P-tubulin, or the tissue-specific PEPCK (Fig. 2).
The translation efficiency of rp mRNAs fluctuates in parallel
to changes in the expression of genes encoding the initiation
factors eIF-4E and eIF-4A. Recently, it has been reported
that the translation of rp mRNAs in reticulocyte extract is
selectively repressed. Supplementation of the extract with
initiation factor eIF-4F or eIF-3, but not other initiation
factors, stimulated the translation of rp mRNAs (27). Consequently, we set out to examine whether the changes in the
efficiency of translation of rp mRNAs that we have observed
in rat liver correlate with alterations in the expression of
initiation factor genes. To this end, we selected two components of the eIF-4F complex, eIF-4E and eIF-4A, and
monitored the relative abundance of the respective mRNAs
during liver development and regeneration. Northern blot
hybridization of liver poly(A)+ mRNA from fetal, adult,
sham-operated, and partially hepatectomized rats revealed
that the levels of these mRNAs follow the same pattern as
does the polysomal association of the rp mRNAs (Fig. 3).

MOL. CELL. BIOL.

a

elF-4E

2 3

4 5

gst

_
_~~~~~~~~V
eIF:-4A

AlbLminr

_m

0at

b
a)

120

co

100-

'

8060-

CD

40-

cu

20

0F

SO

PH

FIG. 3. Changes in the relative abundance of various mRNAs

during liver development and regeneration. (a) Northern blot analysis of poly(A)+ mRNA (2.5 ,ug) from livers of two 18-day fetal
litters (lanes 1 and 2) and from adult (lane 3), sham-operated (lane 4),
and two partially hepatectomized (15-h postoperation (lanes 5 and 6)
rats. RNAs were analyzed by Northern blot hybridization with the
probes indicated at the left. The eIF-4A probe visualizes two mRNA
species (2.0 and 1.6 kb) and the eIF-4E probe visualizes four species
(2.9, 2.5, 1.8, and 1.6 kb), as previously reported (32, 57). (b)
Relative abundance of the various mRNAs determined by densitometric scanning of the most intense bands in the autoradiograms
presented in panel a (the 1.8- and 1.6-kb transcripts of eIF-4E and
eIF-4A, respectively). The filled, stippled, and hatched bars represent the mRNAs of eIF-4A, eIF-4E, and albumin, respectively. The
F (fetal) bars and PH (partially hepatectomized) bars represent
averages of two liver preparations. The densitometric signals of
eIF-4E and eIF-4A were normalized to the average values of the
18-day fetuses, and those of albumin were normalized to the adult
(A) value. The reference values were arbitrarily set at 100. SO,
sham-operated rats.

Thus, the abundance of the mRNAs encoding eIF-4E and

eIF-4A decreased by about 70 and 90%, respectively, upon
development. Similarly, the abundances of these mRNAs
increased by factors of about 2 and 4, respectively, within 15
h after partial hepatectomy. Since the translation efficiency
of eIF-4E mRNA, as judged by its polysomal association,
remains unchanged during liver development and regeneration (50), the synthesis rate of the corresponding protein
should reflect the fluctuations in the abundance of its
mRNA. The apparent 2.5-fold increase in the abundance of
albumin mRNA in the adult rat liver (Fig. 3) is consistent
with that previously reported (72). Hence, the lower mRNA
abundance of both initiation factors in the adult liver seems
to reflect an authentic developmental change rather than

variability in mRNA loading.

The accumulation of rp mRNAs and rRNA during liver
development and regeneration is posttranscriptionally regulated. As mentioned earlier, our results suggest that the
abundance, as well as the translation efficiency, of rp mRNAs decreases upon development. To more closely monitor
the extent and the time course of the developmental changes
in rp mRNA abundance, we extracted total cytoplasmic
RNA from livers of fetal, neonatal, and adult rats and

RIBOSOMAL PROTEIN GENE EXPRESSION IN RAT LIVER

VOL. 12, 1992

b
a

rRNA

L30 a*i
L7

4_

bb

,iw

L18a 0

17d

Actin
Fetal

SW0

oLEEhIU

oil

19d

1I

1Sd

Adult

Age

PEPCK
0
FIG. 4. Ontogeny of the level of various liver mRNAs. (a)
Northern blot analysis of cytoplasmic RNA (12 ,ug) extracted from
livers at days 17 (17d) and 19 (19d) of gestation, at days 1 (1d) and 15
(15d) postpartum, and from adult rats. RNAs were analyzed by
Northern blot hybridization with the probes indicated at the left.
The same blot was sequentially hybridized with L7 and SOD probes.
The ethidium bromide-stained 18S rRNA in the top panel served as
an internal reference standard for comparison of the amounts of
loaded RNAs in the different lanes. (b) Relative abundance of the
various mRNAs determined by densitometric scanning of the autoradiograms presented in panel a. The densitometric signals were
normalized to the value of 17-day fetus for rp mRNAs (presented as
averages + standard errors of the means of the seven mRNAs
shown in panel a) as well as for actin mRNA and to the value of the
adult for PEPCK mRNA. The reference values were arbitrarily set
at 100.

analyzed them by Northern blot hybridization (Fig. 4). ThE

results indicate that the levels of all seven rp mRNAs
examined markedly decreased (sevenfold) from the fetus (17
days) to the adult animal (6 weeks), with a major drop (about
threefold) between the day 19 in utero and day 1 postpartum.
This pattern of changes is almost reciprocal to that observed
with the tissue-specific mRNA encoding liver cytosolic
PEPCK. This mRNA is absent in the fetal liver, as the
transcription of the corresponding gene initiates at birth (Fig.
4) (7). Nevertheless, the decrease in the relative abundance
of rp mRNAs is not unique, as other housekeeping mRNAs,
encoding 1-actin, ,-tubulin, a-tubulin, and the initiation
factors eIF-4E and eIF-4A, are similarly repressed during
liver development (Fig. 3 and 4) (61, 63). It should be noted,
however, that this down-regulation does not involve all
housekeeping mRNAs, as the level of SOD mRNA in the
adult liver, despite developmental fluctuations, is similar to
the fetal level (Fig. 4).
To verify whether the extent to which the observed
alterations in the rp mRNAs are attributable to changes in
transcriptional activity, we carried out nuclear runon transcription assays. Labeled RNA from liver nuclei of 17-day
fetuses and 6 week-old adult rats were hybridized to a set of
filter-bound plasmids that included cDNAs for (i) eight
different r-proteins which, because of the relatively weak

2207

signals of the individual probes, were grouped in two mixtures of four cDNAs each (rpl and rp2), (ii) actin, and (iii)
PEPCK (Fig. 5a). The results indicate that regardless of the
rp probes used, the transcription rate of rp genes does not
change between fetal and adult liver. Thus, the apparent
decline in the abundance of the corresponding mRNAs is
due to a posttranscriptional mechanism. A similar mechanism seems to affect other housekeeping genes, such as the
1-actin and ax-tubulin genes, in the developing liver (Fig. 5a)
(61, 63). Evidently, these nuclear preparations are normal in
the sense that they exhibit the typical developmental regulation of the transcription of cytosolic PEPCK gene (Fig. 5a)
(8).
When the densitometric signals are corrected for the
number of thymidine residues in the cDNA probes (574, 371,
and 427 for rpl, rp2, and PEPCK cDNAs, respectively), the
rate of transcription of the tissue-specific gene encoding
PEPCK in the adult liver is eightfold higher than that of the
rp genes. Furthermore, the weaker autoradiographic signal
obtained with the rp2 probe mixture (6.5 densitometric
units), than with that obtained with rpl (35 densitometric
units) apparently reflects the lower overall number of uridine
residues in the rp2 cDNA mixture, as well as the lesser
resistance to the RNase A wash, as a result of mismatches
between the rat transcripts and the heterologous cDNAs in
the rp2 mixture.
The coordinated accumulation of r-proteins and rRNA has
been observed in a variety of cells stimulated to proliferate,
yet uncoupling of the synthesis of these two classes of
molecules has been documented in several growth-arrested
systems (reviewed in reference 49). To examine whether the
repression of rRNA is in accordance with the decrease in the
abundance of the rp mRNAs, we monitored the relative
transcription rate of rDNA in nuclei isolated from fetal and
adult liver (Fig. Sb). These measurements were carried out
either with untreated nuclei and a probe (pB5) containing
mostly 18S sequences (experiment 1) or with RNase
A-treated nuclei (to eliminate unlabeled rRNA, which might
compete with the 32P-labeled RNA in the hybridization
reaction) and a probe (plla.2) containing mostly 28S sequences (experiment 2). The apparently similar results obtained in these two experiments suggest that the synthesis of
rRNA is not inhibited during the development of the liver.
To elucidate the level at which rp gene expression is
induced in the regenerating liver, we measured the relative
transcription rates of the corresponding genes. Runon transcription assay was performed with nuclei from livers of
control rats or 12 h after partial hepatectomy or sham
operation (Fig. 5c). The results obtained with either a
mixture of six rp probes or only the rpL18a probe indicate
that changes in the rates of transcription of the respective
genes are undetectable prior to or at the peak accumulation
of rp mRNAs. Consequently, it appears that the elevation in
the abundance of rp mRNAs during liver regeneration results from a posttranscriptional mechanism, as in the devel-

oping liver.
Nuclei prepared from livers of partially hepatectomized
and sham-operated rats were used to monitor the transcriptional activity of rDNA at various times after the operation
(Fig. Sd). The results demonstrate an increase in the relative
transcription rate of the rDNA (about 2.5-fold at 12 h
postoperation), both the extent and time course of which are
consistent with those previously obtained by using either
isolated nuclei (58) or pulse-labeled whole animals (15).
The decrease in the steady-state level of rRNA during liver
development is similar to the decline in the translation effi-

2208

ALONI ET AL.
a

MOL. CELL. BIOL.

120

140
120

100

O.o

80
0
C

60

60

' 40

40

20

20

0aa
co

Expl

Exp 2

4)

0
0..
0
C
U
0

C)
0
O-

Control

SO (12h) PH (12h)

12

18

Time (hrs)

FIG. 5. Relative transcription rates of various ribosome-associated genes during development and regeneration of rat liver. (a) The relative
transcription rates of rp genes during development. Nuclei were isolated from fetal (17 days in utero) and adult (6 weeks) livers. Nascent RNA
molecules were elongated in 2 x 107 isolated nuclei in the presence of [32P]UTP. In two independent experiments with essentially the same
results, the average levels of incorporation by fetal and adult nuclei were 2.2 x 107 and 1.8 x 107 cpm, respectively. Equal amounts of labeled
RNA (about 8 x 106 cpm) were hybridized to filter-bound probes (see Materials and Methods). rpl, a mixture of rat cloned rp cDNAs
corresponding to L5, L7, L18a, and L19; rp2, a mixture of cloned rp cDNAs corresponding to mouse L30, L32, and S16 as well as human
P2 cDNA; actin, mouse cloned a-actin cDNA; PEPCK, rat cloned PEPCK cDNA. The results are presented in densitometric units and
represent two independent experiments with similar results. The transcription rates of the genes denoted by rpl, rp2, and actin in the adult
were calculated relative to that in the fetus. In contrast, the transcription rate of the PEPCK gene in the fetus was normalized to that in the
adult. The absolute densitometric signals (arbitrary units) for rpl, rp2, actin, and PEPCK are 35, 6.5, 3.5, and 210, respectively. (b) Relative
transcription rate of rDNA during rat liver development, measured essentially as described for panel a. The probes used in experiment 1 (Exp
1) and experiment 2 (Exp 2) were pB5 and plla.2, respectively. The nuclei in experiment 2 were treated with RNase A during their preparation
(67). In both experiments, series of filter-bound DNAs were simultaneously hybridized with 105, 2 x 105, 4 x 105, and 8 x 105 cpm of
32P-labeled RNA. The average density of the autoradiographic signal per input of 105 cpm was calculated after scanning the different
autoradiograms of each series. The transcriptional rate of rDNA in nuclei from adult liver was normalized to that of fetal liver. (c) Relative
transcription rates of rp genes in regenerating rat liver. Liver nuclei from intact animals (control), sham operated (SO), and partially
hepatectomized (PH) rats were isolated 12 h postoperation. The runon transcription assay was performed with 10 nuclei, with average levels
of incorporation of 4.2 x 107, 3.5 x 107, and 4.1 x 107 cpm for control, sham-operated, and partially hepatectomized animals, respectively.
32P-labeled RNA (1 x 107 to 2 x 107 cpm) was hybridized with a filter-bound mixture (3 pug each) of six plasmids bearing cDNAs encoding
rat rpL7, rpL18a, and rpL19 and mouse rpL30, rpL32, and rpS16 or only rpL18a cDNA. The densitometric signals were corrected for both
background and differences in the [32P]RNA input in the hybridization reaction. The transcription rate in operated animals was calculated
relative to that in control animals. Each bar represents the average + standard error of the mean of results obtained with nuclei from three
different animals. (d) Time course of the effect of partial hepatectomy on the relative transcription rate of rDNA. Nuclei were isolated from
partially hepatectomized (PH) and sham-operated (SO) rats at the indicated time postoperation and from an intact animal (dashed line).
Elongation of nascent RNA chains was carried out with 4 x 106 nuclei, and filter-bound plla.2 DNA was hybridized with 2 x 105 cpm of
32P-labeled RNA. The relative transcription rate was normalized for the value from an intact animal (zero time). In all experiments presented
in panels a to d, filters were extensively washed following hybridization and then autoradiographed. The autoradiographic images were
quantified by densitometric scanning, with subtraction of background values obtained with a vector blank. The reference values were
arbitrarily set at 100.

ciency of rp mRNAs. The reduction in both the abundance

(sevenfold) and the translation efficiency (about threefold) of
the rp mRNAs should potentially lead to an overall diminution in the synthesis capacity of r-proteins by more than an
order of magnitude. To assess whether the abundance of
ribosomes in adult liver proportionally decreases, we monitored the change in steady-state level of liver 28S rRNA
between fetal and adult life. Liver samples from three litters
of 18-day in utero and three adult rats were separately
homogenized, total cytoplasmic RNA was extracted, and the
poly(A)+ mRNA was removed by oligo(dT) column chroma-

tography. The poly(A)- RNA was size fractionated on a

sucrose gradient, and the 28S rRNA was isolated and
quantified by optical density at 260 nm. The recovery (65 to
100%) of the 28S rRNA was estimated by inclusion, throughout the purification procedure, of 3H-labeled 28S rRNA
(55,000 cpm/15.2,ug), similarly isolated from Ltk- cells,
labeled with [3H]uridine. The results indicate that the total
amount of steady-state 28S rRNA declined just twofold,
from 4,343 96,ug per g of fetal liver to 2,109 + 354,ug per
g of adult liver. This decrease is consistent with that observed for the translation efficiency of rp mRNAs in the

RIBOSOMAL PROTEIN GENE EXPRESSION IN RAT LIVER

VOL. 12, 1992

developing liver and is similar to that reported for NIH 3T3

fibroblasts (74).
DISCUSSION
Translational control of rp gene expression during liver
development and regeneration. Our results provide the first
evidence that translational control of mammalian rp mRNAs
is not confined to manipulated cultured cells but operates
also in the course of a normal liver development. Thus, when
liver cells become quiescent in the adult animal, the rp
mRNAs are translationally repressed. Nonetheless, this
change is reversible, as resumption of cellular growth,
following partial hepatectomy, elicits a three- to fourfold
increase in the translation efficiency of rp mRNAs relative to
that of untreated adult animals (Fig. lb and 2). The extent of
this increase, in conjunction with the two- to threefold
elevation in the abundance of these mRNAs (20, 56), fully
accounts for the increase (about 10-fold) in the synthesis rate
of the r-proteins under these circumstances (73). It appears,
therefore, that the extent of association of rp mRNAs with
polysomes is a valid measure of their translational activity.
This conclusion has been further corroborated in our recent
study of the translation efficiency of chimeric rp-human
growth hormone mRNAs. Our results have indicated that
the degree of repressed translation in the nongrowing cells,
as measured by the polysome assay, is similar to that
directly assessed by an immunoassay of the final protein
product.
Notably, the regenerating liver represents the only known
physiological condition under which the rp mRNAs are
almost fully engaged in translation, as indicated by the
proportion (over 90%) of their association with polysomes
(Fig. lb and 2). This is in sharp contrast to cultured cells, in
which rp mRNAs are always underutilized, even under
conditions of extensive proliferation (2, 35, 51, 53).
Liver development is associated with an enrichment of
parenchymal tissue at the expense of hematopoietic elements (24). It appears, however, that in the adult liver, the
decrease in both the abundance and the translation efficiency
of rp mRNAs reflects transition into a resting state rather
than altered cellular composition. This conclusion can be
inferred from the opposite changes, induced by partial
hepatectomy, as they occur prior to the first mitosis and
therefore cannot result from an alteration in cellular populations.
It should be noted that expression in the rat liver of the
housekeeping genes encoding the heavy and light chains of
ferritin is also regulated at the translational level (6). This
translational control involves a specific protein which binds
to a stem-loop structure in the 5' untranslated region of
ferritin mRNA and represses its translation (37). However,
unlike the translation of ferritin mRNA, which is responsive
to the intracellular iron concentrations, that of rp mRNAs
specifically responds to the cellular growth status.
In an attempt to delimit the translational regulatory element (TLRE) within the rp mRNAs, we have previously
shown that as few as 29 5'-terminal nucleotides of mouse rp
S16 mRNA are sufficient to confer translational repression
on a chimeric mRNA in growth-arrested mouse lymphosarcoma cells (41). The involvement of a similar TLRE in the
translational control of rp mRNAs has been established also
during myoblast differentiation (27) and Xenopus development (44). Furthermore, we have demonstrated that the
5'-terminal pyrimidine tract, starting with a cytidine residue

2209

at the initiating residue, plays a critical role in this translational control mechanism (41).
The putative trans-acting factors that determine the
growth dependent activity of the TLRE have not yet been
identified. Nevertheless, one can hypothesize that the translational control may involve a general translational initiation
factor. If such a factor had a particular low affinity for rp
mRNAs, because of their unique 5' terminus, a decrease in
its activity or content could lead to a selective diminution in
the translation of these mRNAs. A prime candidate is the
eIF-4F complex, which has been implicated in discrimination between weak and strong mRNAs and consists of three
subunits: eIF-4E (the cap-binding protein), eIF-4A, and a
220-kDa protein (64, 70). Because of the relatively low
abundance of eIF-4E (18), the entire eIF-4F complex is a
limiting component in the binding of eukaryotic mRNAs to
the ribosome. Since this step is generally considered to be
the overall limiting step in translation (76), regulation of
translation efficiency of the rp mRNAs might involve modulation of the activity or the amount of eIF-4F. This notion
is supported by (i) the correlation between the changes in the
polysomal association of the rp mRNAs during liver development and regeneration and the alterations in the abundance of the mRNAs encoding the initiation factors eIF-4E
and eIF-4A (Fig. 3) and (ii) the stimulation, by the addition of
eIF-4F or eIF-3 to reticulocyte extracts, of the translation of
rp mRNAs, which otherwise are selectively repressed (27).
It is noteworthy that the adult liver, although mitotically
inactive, maintains an intensive protein synthetic activity,
which is comparable with that of the fetal liver, as exemplified by their similar polysomal profiles (Fig. la). This is in
sharp contrast to quiescent cells in culture, which exhibit a
significantly lower proportion of ribosomes engaged in translation (35, 51, 53). Hence, it is unlikely that the adult liver is
significantly deprived of initiation factors, but perhaps it
compensates for the lower abundance of the respective
mRNAs by a greater stability of the corresponding proteins.
Alternatively, since phosphorylation of eIF-4E has been
implicated in regulation of translation efficiency (18, 35, 67,
70), the relatively unaltered protein synthetic capacity in the
adult rat liver might be a result of a higher proportion of this
factor in its phosphorylated form.
Clearly, even a minor decrease in the activity of general
initiation factors might impede the translation of a subset of
mRNAs. However, it appears that the changes in the levels
of eIF-4E and eIF-4A and/or the respective mRNAs cannot
fully account for the translational control of rp mRNAs.
Thus, translation efficiency of these mRNAs reaches its
maximal capacity in regenerating liver, prior to complete
recovery of the abundance of the mRNAs for the initiation
factors (Fig. 3). This argument is further supported by the
unique translational control of Xenopus rp mRNAs. These
mRNAs not only are characterized by similar TLREs but
also are subject to translational repression during early
embryogenesis. However, unlike the repressed translation
of rp mRNAs in resting mammalian cells, that of the Xenopus mRNAs occurs during developmental stages characterized by both rapid proliferation and extensive protein synthesis (4). It is likely, therefore, that in addition to involving
a general initiation factor(s), the translational control of rp
mRNAs in both species might involve a TLRE-specific
factor. Conceivably, the TLRE reacts with a specific factor,
which is quantitatively regulated or qualitatively modified in
response to changes in cellular growth rate. Presumably,
binding of this factor to the TLRE prevents or enhances the
interaction of rp mRNAs with ribosomes and other compo-

2210

ALONI ET AL.

nents of the translational machinery. The exceptionally high

efficiency of translation of rp mRNA observed in the regenerating liver might be due to a transient accumulation or
activation of a specific activator or, alternatively, depletion
or inactivation of a specific repressor.
Posttranscriptional regulation of rp gene expression. Our
data indicate that the steady-state level of rp mRNAs is
posttranscriptionally regulated during liver development and
regeneration. The developmental decrease in the abundance
of rp mRNAs has been previously reported for two fortuitously identified mammalian rp mRNAs encoding a rat
r-protein similar to L-3 of Escherichia coli (60) and a mouse
r-protein similar to yeast S4 (3, 28). Nonetheless, unlike the
selective translational control of rp mRNAs, the reduction in
their abundance during liver development seems to be a part
of a general inhibitory effect, which includes many other
housekeeping mRNAs. The SOD mRNA seems to be exceptional by its similar abundances in both fetal and adult liver.
The increase in the abundance of rp mRNAs during liver
regeneration, like the decrease that occurs during development, is not specific, as the abundance of other housekeeping mRNAs, encoding a-tubulin and ,-tubulin, is also elevated through a posttranscriptional mechanism (22). It is
worth noting however, that such dramatic fluctuations in the
abundance of rp mRNAs is unique to the developing liver.
Thus, in all other examined cases of reversible growth arrest
in mammalian cells exhibiting a similar selective translational control of rp mRNAs, the abundance of these mRNAs
remained unchanged (51, 53).
Decrease in ribosome accumulation during development.
The apparent twofold decrease in the steady-state level of

rRNA between fetal and adult liver is consistent with a

previous estimation based on an analytical ultracentrifugation of free ribosomes (59). This change is comparable with

the two- to threefold increase in cellular content of ribosomes, observed during transitions between resting and
growing states of cells in culture (9, 33, 71), which results
from a combination of two or more mechanisms operating in
different cell lines and under various growth conditions.
These include a decline in the rate of rRNA turnover (1, 19,
48, 77), increase in the rate of its synthesis (26, 45), and
increase in the rate of its processing (34, 79), as well as an
increase in the synthesis rate of r-proteins (74).
The r-protein synthesis capacity declines about 20-fold
during liver development as result of a decrease in both the
abundance (7-fold) and translation efficiency (3-fold) rp
mRNAs. Surprisingly, this drop exceeds by an order of
magnitude the decrease in the ribosome content. One plausible explanation for this discrepancy is a possible uncoordinated synthesis of rRNA and r-protein in utero. Uncoupling of these two processes has been previously observed
when rat myoblasts are induced to differentiate into myotubes, in which the synthesis of r-proteins remains constant
while that of rRNA decrease 5- to 10-fold (30, 38). If a similar
situation is applicable for fetal liver, then the production of
r-proteins exceeds that of rRNAs and unassembled r-proteins are readily degraded.
Synthesis of rRNA in regenerating liver is induced by an
increased transcription of the rDNA. Removal of two-thirds
of rat liver leads to about a 10-fold increase in the synthesis
rate of rRNA (14, 73). This elevation results from both an
approximately 2.5-fold increase in the transcription rate of
the rDNA (Fig. 5d) (15, 58) and a similar increase in the rate
of the posttranscriptional events, including pre-rRNA processing and nuclear-cytoplasmic transport (16). It appears,
therefore, that the liver cells exploit different regulatory

MOL. CELL. BIOL.

mechanisms to control the abundance of rRNA during

development (posttranscription) and regeneration (transcription and posttranscription). Possibly, the acute response
following partial hepatectomy requires recruitment of both
mechanisms, whereas a posttranscriptional mechanism is
sufficient to cope with the slow changes in growth rate which
occur during development. Interestingly, partial hepatectomy of the Rku: NCS (S) specific-pathogen-free mouse
strain, unlike that of rats of various origins (14, 73; this
report), did not stimulate accumulation of rp mRNAs or
increase in the synthesis rate of rRNA (22). Similar species
specific differences have been previously demonstrated for
the regulation or rp gene expression during mouse and rat
myoblast differentiation (2, 30).
ACKNOWLEDGMENTS
We thank N. Sonenberg for the mouse eIF-4E cDNA clone, P.
Nielsen for the mouse eIF-4A cDNA clone, I. Ginzburg for the rat
,-tubulin cDNA clone, and Y. Groner for the human SOD cDNA
clone.
This research was supported by grant 86-0070 from the United
State-Israel Binational Science Foundation, Jerusalem, Israel, and
by a grant from the Israel Cancer Research Fund to O.M.

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