Báo cáo khoa học: Synthesis and function of ribosomal proteins – fading models and new perspectives pptx

REVIEW ARTICLE
Synthesis and function of ribosomal proteins – fading
models and new perspectives
Sara Caldarola, Maria Chiara De Stefano, Francesco Amaldi and Fabrizio Loreni
Department of Biology, University ‘Tor Vergata’, Roma, Italy
Introduction
Ribosomal proteins (RPs) are fundamental compo-
nents of ribosomes. They assemble with four rRNA
molecules in a complex process that takes place
sequentially in the nucleolus, in the nucleoplasm, and
in the cytoplasm. Nearly 200 nonribosomal factors are
required for the synthesis, maturation and export of
the two ribosomal subunits [1]. Most of the constitu-
ents of the preribosomal particles have been identified
in yeast by exploiting the potent combination of
genetic and biochemical approaches [2]. More recently,
advances in MS techniques have also led to the identi-
fication of the nucleolar proteome in human cells [3].
The role of RPs in the assembly of ribosomes has been
studied for many years. Reconstitution experiments in
prokaryotes have shown a specific order of addition of
RPs for self-assembly of ribosomal subunits [4,5]. The
greater complexity in the assembly of the eukaryotic
ribosome has until now prevented in vitro reconstitu-
tion. However, a recent analysis of the in vivo assembly
pathway of the 40S ribosomal subunit showed that the
formation of distinct structural intermediates may be
similar to what occurs in the prokaryotic counterpart
[6]. The structure and function of the ribosome appear
to be generally conserved in all organisms. The small
subunit (30S or 40S) contains the decoding center,

whereas the large subunit (50S or 60S) is responsible
for the catalysis of the peptide bond formation, due pri-
marily to rRNA. However, the initiation, termination
Keywords
mTOR signaling; nucleolus; protein
synthesis; protein turnover; ribosomal
pathology; ribosomal stress; ribosome
biogenesis; TOP mRNA; translational control
Correspondence
F. Loreni, Department of Biology, University
‘Tor Vergata’, Via Ricerca Scientifica, 00133
Roma, Italy
Fax: +39 062023500
Tel: +39 0672594317
E-mail:
(Received 16 February 2009, revised 18
March 2009, accepted 2 April 2009)
doi:10.1111/j.1742-4658.2009.07036.x
The synthesis of ribosomal proteins (RPs) has long been known to be a
process strongly linked to the growth status of the cell. In vertebrates, this
coordination is dependent on RP mRNA translational efficiency, which
changes according to physiological circumstances. Despite many years of
investigation, the trans-acting factors and the signaling pathways involved
in this regulation are still elusive. At the same time, however, new tech-
niques and classic approaches have opened up new perspectives as regards
RP regulation and function. In fact, the proteasome seems to play a crucial
and unpredicted role in regulating the availability of RPs for subunit
assembly. In addition, the study of human ribosomal pathologies and
animal models for these diseases has revealed that perturbation in the syn-
thesis and ⁄ or function of an RP activates a p53-dependent stress response.

Surprisingly, the effect of the ribosomal stress is more dramatic in specific
physiological processes: hemopoiesis in humans, and pigmentation in mice.
Moreover, alteration of each RP impacts differently on the development of
an organism.
Abbreviations
Atg7, autophagy-related gene 7; CNBP, cellular nucleic acid-binding protein; DBA, Diamond–Blackfan anemia; E, embryonic day; eIF,
eukaryotic initiation factor; mTOR, mammalian target of rapamycin; NEDD8, neural-precursor-cell-expressed developmentally downregulated
8; PI3K, phosphoinositide-3-kinase; RP, ribosomal protein; S6K, S6 kinase; TOP, terminal oligopyrimidine; TSC, tuberous sclerosis complex;
TSS, transcription start site; USP10, ubiquitin-specific protease 10; ZNF9, zinc finger protein 9.
FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS 3199
and recycling phases of translation show differences
between prokaryotic and eukaryotic ribosomes [7].
Consistent with this observation, there are differences
in the protein composition of the ribosomes from the
different kingdoms. Of the 80 mammalian RPs, 49 are
related to archeal RPs, and 32 are homologous to bac-
terial proteins [8]. The remaining 11 RPs are specific
for eukaryotic ribosomes and may be involved in addi-
tional particular functions, such as intracellular trans-
port. Alternatively, they may be required for the more
complex regulation of eukaryotic protein synthesis [9].
The identification of the role of a specific RP is com-
plicated by the high level of cooperativity among ribo-
somal components and by the fact that the ribosome is
essential for the cell. Accordingly, most of the analyzed
eukaryotic RPs have been reported as being essential
for growth [9,10]. It can be postulated that RPs are
required for different steps of ribosome biogenesis
and ⁄ or ribosome function. Indeed, a systematic study
of the incorporation of RPs into preribosomes led to

the identification of the in vivo assembly pathway of
the eukaryotic small ribosomal subunit [6]. In some
cases, specific RPs have been shown to play a role in
ribosomal functions such as interaction with transla-
tion initiation factors, translation accuracy, and pep-
tide bond formation [9,11]. Although trans-acting
factors involved in ribosome biogenesis as well as pre-
rRNA processing are well conserved among eukary-
otes, the synthesis of RPs appears to be regulated quite
differently in yeast and in mammals. In fact, the more
than 130 yeast RP genes behave as a precisely coordi-
nated transcriptional cluster under a variety of envi-
ronmental conditions [12]. This is because almost all
RP gene promoters in Saccharomyces cerevisiae con-
tain one or two sites for the factor Rap1 [13]. Tran-
scriptional activation or repression is obtained through
the Rap1-dependent recruitment of different additional
factors that combine to determine the correct level of
transcription [14]. By contrast, early studies of mam-
malian RP gene promoters showed that there are no
shared elements, but transcriptional activity is approxi-
mately equivalent [15]. More recent in silico analyses
found some recurring motifs in the transcriptional con-
trol regions [16,17]. However, besides some variation
of RP transcript levels in different tissues and in neu-
ronal differentiation [18,19], transcriptional regulation
does not appear to play a major role in the control of
RP synthesis. In fact, it is now well documented that
there are signaling pathways that regulate the transla-
tional activity of RP mRNA for adjustment of the

biosynthesis of ribosomes to the requirements of cell
growth and differentiation. In addition, a relevant
contribution of protein turnover to the regulation of
RP synthesis and accumulation has been proposed by
recent studies [20]. Therefore, this review will focus on
the different aspects of translational and post-transla-
tional regulation of RP metabolism. We will also high-
light the role that studies on putative ribosome
pathologies have had in our understanding of regula-
tory mechanisms of RP synthesis.
Translational regulation of RP
synthesis
Sequence comparison of some vertebrate RP genes
cloned in the early 1980s revealed that these genes
share a characteristic and distinctive structure of the
transcription start site (TSS), which is always posi-
tioned within a pyrimidine stretch (about 10–25 nucle-
otides long), so that the transcribed mRNAs always
start with a C followed by a stretch of 5–15 pyrimi-
dines. Later, it was found that this TSS structure char-
acterizes all vertebrate RP genes, including all of the
80 human RP genes. This structure is rather peculiar,
given that the vast majority of mRNAs start with a
purine, most often an A. There are a number of other
genes, implicated directly or indirectly in translation,
that share this peculiar TSS structure. Among these,
we find all the translation elongation factors, but only
a few of the numerous translation initiation factors,
i.e. eukaryotic initiation factor (eIF) 3e, eIF3f, and
eIF3h [21].

The genes whose corresponding mRNAs begin with
a5¢-terminal oligopyrimidine (TOP) sequence and are
translationally regulated have been named ‘TOP
genes’. In fact, external signals, such as stress or the
availability of growth factors, hormones, and nutrients,
result in the activation of signaling pathways that rap-
idly and reversibly modulate the translation of RP
mRNAs and the other TOP mRNAs (Fig. 1).
It has been observed that, besides the TOP sequence,
RP genes are characterized by short UTRs. For
instance the 5¢-UTRs of the 80 human RP mRNAs
have an average length of 40 nuceotides (range
12–125), which is rather shorter than the average
human 5¢-UTR. Even more striking are the 3¢-UTRs,
which, in the 80 human RP mRNAs, have an average
length of 35 nucleotides, in contrast to almost 1000
nucleotides for the average human 3¢-UTR.
To understand the molecular mechanism involved in
the growth-associated translational regulation of RP
mRNAs, several studies have sought to identify the
cis-acting elements and the trans-acting factors that
might be responsible for this specific control.
Studies on various vertebrate systems (Xenopus,
mouse, and human) have amply demonstrated that the
Ribosomal protein synthesis S. Caldarola et al.
3200 FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS
TOP sequence present at the 5¢-end of all RP mRNAs
represents the major cis-acting element [22]. However,
the 3¢-UTR of RP mRNA may also play a role in
translational regulation. In fact, although this short

region does not confer translational regulation on a
reporter mRNA without the TOP sequence, it does
contribute to the stringency of the regulation of a TOP
containing RP mRNA [23].
Putative trans-acting factor(s) that might be involved
in the growth-dependent translational regulation of RP
mRNAs have remained more elusive. In Xenopus , two
proteins have been identified, La and cellular nucleic
acid-binding protein (CNBP) ⁄ zinc finger protein 9
(ZNF9), which bind the 5¢-UTRs of RP mRNAs in vitro.
La interacts with the TOP sequence, whereas CNBP
binds a sequence element located closely downstream
[24,25]. The mutually exclusive binding of these two
proteins on the 5¢-UTRs of TOP mRNAs led
Pellizzoni to propose that La may increase translation,
whereas CNBP ⁄ ZNF9 could act as a translational
repressor. The interaction of La with RP mRNA has
also been confirmed in human cells, where La has been
shown to exist in two distinct states that differ in sub-
cellular localization [26]. When La is phosphorylated
on serine 366, it is localized in the nucleus, where it
has a role in polymerase III gene transcription. In con-
trast, nonphosphorylated La is found in the cytoplasm,
where it binds TOP mRNAs. Moreover, immunocom-
plex precipitation of La from HeLa cellular extracts
yields a number of mRNAs, including TOP mRNAs,
thus supporting the conclusion that La protein binds
TOP mRNAs in vivo. More recently, it has been shown
that La can also be phosphorylated by AKT, which is
a component of a signaling pathway involved in TOP

mRNA regulation [27] (see below). Several studies
have been set up to verify whether La is actually impli-
cated in translational regulation. Unfortunately, the
results lack coherence, and make it difficult to draw a
Fig. 1. Synthesis and turnover of ribosomes. RPs are translated into the cytoplasm and imported into the nucleolus, where they are
degraded by proteasomes or assembled with rRNAs into ribosomal subunits. 40S and 60S subunits are then exported into the cytoplasm to
form mature ribosomes that are able to initiate translation or are degraded via autophagy, probably through the USP10–G3BP1 complex.
S. Caldarola et al. Ribosomal protein synthesis
FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS 3201
final conclusion. For instance, inducible overexpression
of La in stably transfected Xenopus cell lines had a
positive effect on translation of RP mRNAs [28]. This
is consistent with the positive role of La observed in
the internal ribosome entry site-mediated translation of
picornaviruses [29]. On the other hand, opposite results
were reported by Schwartz et al. [26]. In addition,
recent experiments carried out in our laboratory in
human cells showed that neither La overexpression nor
downregulation by RNA interference had any signifi-
cant effect on translation of RP mRNAs (M. C. De
Stefano, unpublished results). Finally, the binding of
La to a chimeric human TOP containing 5¢-UTR
reporter mRNA inhibits its translation in vitro [30].
Similarly, experiments on CNBP ⁄ ZNF9 showed that
overexpression of this protein can inhibit the transla-
tion of a chimeric TOP–green fluorescent protein
mRNA, but that its downregulation by RNA interfer-
ence does not interfere with the growth-associated
translational activity of TOP messengers (S. Caldarola,
unpublished results). A possible explanation for the

inconsistencies could be that additional factors contrib-
ute to the regulation. For instance, Ro60 is known to
interact with La and CNBP ⁄ ZNF9, whereas small
RNAs (Y) form a complex with La. If all of these
factors play a role in the regulation, the overexpression
or downregulation of only one of them could produce
apparently contradictory results in different experimen-
tal systems and conditions. A different situation is pre-
sented in a recent report by Orom et al. These authors
indicate microRNA-10a to be a trans-acting element
implicated in the translational regulation of RP
mRNAs [31]. The pairing of microRNA-10a with
the 5¢-UTRs of three RP mRNAs stimulates RP
mRNA translation. This mechanism is unusual for
microRNAs because, in general, they have a negative
effect on mRNA translation by interacting with their
3¢-UTRs [32], and it is not known whether it can be
extended to other TOP mRNAs.
Signaling pathways to RP mRNA
translation
As most of the reports addressing signaling consider
TOP mRNA as a homogeneous group, in this section
we will refer to RP mRNA as TOP mRNA. In the last
15 years, various research groups have studied the sig-
nal transduction pathways involved in TOP mRNA
translational control. Polysome separation on sucrose
gradients, which allows analysis of the polysome ⁄ sub-
polysome distribution of a messenger, has been used to
monitor the translation efficiency of TOP messengers
in different growth conditions. A variety of signals,

such as stress or the availability of growth factors,
hormones, and nutrients, can induce a change in the
percentage of TOP mRNA associated with polysomes
from 30–40% to 65–75%, and vice versa [33]. Several
lines of evidence converge in indicating phosphoinosi-
tide-3-kinase (PI3K) as a key modulator of TOP
mRNA translation after mitogenic stimulation [34].
PI3K activates a signaling pathway that includes:
phosphoinositide-dependent kinase 1, protein kinase B
(also called AKT), tuberous sclerosis complex (TSC)1–
TSC2, and the mammalian target of rapamycin
(mTOR) C1 complex (composed of raptor, mLst8, and
mTOR). The role of TSC1–TSC2 in the translation of
TOP mRNAs has been recently investigated by Bilanges
et al. [35], using microarray analysis. The authors
analyzed the translational efficiency of many cellular
messengers in wild-type, TSC1
) ⁄ )
or TSC2
) ⁄ )
mouse
embryo fibroblasts, during serum starvation and ⁄ or
treatment with the mTORC1 inhibitor rapamycin.
They found that translation of most TOP mRNAs is
regulated by mitogen-induced signal transduction path-
ways acting through TSC1–TSC2 and involving
mTORC1, as suggested by the rapamycin effect.
Rapamycin, which inhibits mTORC1 by binding to
mTOR in a complex with the immunophilin FKBP12,
has a variable effect on TOP mRNA translation. In

HeLa cells, it totally blocks the recruitment of TOP
messengers on polysomes during serum stimulation
[33]. In other cell lines, however, this inhibitory effect
is only partial [34,36]. Recent data from the Meyuhas
group indicate that mTOR is indispensable for the
translational activation of TOP mRNAs [37]. How-
ever, these authors showed that decreasing the expres-
sion of the raptor or rictor genes (partners of
mTORC1 and mTORC2 respectively) has only a slight
effect on the translation efficiency of TOP mRNAs.
This result implies that mTOR regulates TOP mRNA
translation through a novel rapamycin-insensitive
pathway with a minor, if any, contribution of the
canonical mTOR complexes mTORC1 and mTORC2.
A further downstream target of the PI3K pathway is
RPS6, which is phosphorylated after mitogenic stimu-
lation by two closely related kinases, S6 kinase (S6K)
1 and S6K2. The strong correlation between the trans-
lational activation of TOP mRNAs and the hyper-
phosphorylation of RPS6 [36] led to the assumption
that RPS6 phosphorylation was necessary for the
recruitment of TOP messengers to the polysomes [38].
For years, RPS6 has been considered to be the key
protein responsible for the selective translation of TOP
mRNAs able to increase the affinity of ribosomes
for this class of messengers. However, this model was
initially questioned by the observation that in cells
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3202 FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS
from S6K1

) ⁄ )
⁄ S6K2
) ⁄ )
mice, the translation of
TOP mRNAs is still modulated by mitogens in a rapa-
mycin-dependent manner [39]. Unexpectedly, RPS6
phosphorylation at serine 235 and serine 236 persisted
in the absence of both S6K1 and S6K2, revealing the
presence of another S6K, most likely p90 ribosomal S6
kinase. More recently, to abolish any residual phos-
phorylation on RPS6, Meyuhas et al. produced a via-
ble and fertile knock-in mouse with mutated
unphosphorylatable RPS6 (RPS6P
) ⁄ )
). Mouse embryo
fibroblasts isolated from RPS6P
) ⁄ )
mice still show
serum-dependent translational activation of TOP mes-
sengers. This indicates that complete abrogation of
RPS6 phosphorylation does not affect the translation
of TOP mRNAs, definitely disproving the model [40]
(shown schematically in Fig. 2).
Although TOP mRNAs have been generally consid-
ered to be a homogeneous group regulated in a coordi-
nated way, a recent report from Sonenberg’s
laboratory identified a subset of TOP mRNAs whose
translation is influenced by eIF4E overexpression [41].
eIF4E is the limiting component of the eIF4F initia-
tion complex, and a key player in the regulation of

translation in eukaryotic cells. It is thought to enhance
the translation of mRNAs with highly structured
5¢-UTRs [42], and to play an important role in cell
growth and proliferation [43,44]. Moreover, eIF4E is
overexpressed in many kinds of cancer, and its abun-
dance is correlated with the progression of malignan-
cies [45]. In order to identify messengers regulated by
eIF4E, Sonenberg et al. performed a microarray analy-
sis of polysome-associated mRNAs from NIH3T3 cells
overexpressing eIF4E. They identified messengers cod-
ing for proteins involved in cell proliferation (MIF and
cenpA), survival (i.e. survivin, BI-1, and dad1), and
ribosome biogenesis (members of the small and large
ribosomal subunits). Interestingly, not all RP mRNAs
respond to eIF4E overexpression, suggesting the
existence of subclasses of TOP mRNAs with different
regulatory mechanisms.
RP turnover
Ribosome production is strongly linked to the rate of
cellular growth. The construction of ribosomes is
among the most energy-consuming events that occur in
a cell. A growing HeLa cell synthesizes about 7500
ribosomal subunits per minute, using up some 300 000
RPs, accounting for almost 50% of all cellular proteins
in growing cells [46]. Mature ribosomes are very stable
complexes, with an estimated half-life of about 5 days
for both RPs and rRNA [47]. Several laboratories have
tried to understand how ribosomes are recycled and
whether there is a specific mechanism of degradation to
adjust their number. In a recent report by the Andersen

group [20], quantitative analyses of RP trafficking in
HeLa cells revealed a prominent role for the protea-
some in regulating their turnover. Using fluorescence
recovery after photobleaching and MS analysis, the
researchers measured the turnover of RPs within the
cell, and observed that newly produced RPs accumulate
in the nucleolus much faster than do other nucleolar
proteins. Despite this, only about one-quarter of the
synthesized RPs are assembled into ribosomes and
exported to the cytoplasm, a large number of them
being degraded via proteasomes. These results indicate
that the nuclear export of RPs assembled in ribosomal
subunits is slower than the import of free RPs, and that
most RPs are produced in excess with respect to the
amount needed for ribosome production. Thus, degra-
dation of nucleolar RPs could be a general mechanism
by which mammalian cells control ribosome produc-
tion, adjusting it according to cellular needs. It has
been observed that ribosomes are abundantly ubiquiti-
nated, suggesting a role of the proteasome in RP turn-
over. Ubiquitination occurs on RPS2, RPS3, RPS20
[48], and RPL27a [49]. The last of these modifications,
identified in HEK293 cells, is conserved in the yeast
homolog L28. RPL27a ubiquitination is reversible and
Fig. 2. Signal transduction pathways involved in TOP mRNA trans-
lational control. Black arrows indicate activation, and bars indicate
inhibition. Gray arrows refer to signaling pathways not involved in
TOP mRNA translational control (see text for details).
S. Caldarola et al. Ribosomal protein synthesis
FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS 3203

cell cycle-regulated, and increases the translational effi-
ciency of ribosomes, indicating that addition of ubiqu-
itin molecules to RPs can also have a nonproteolytic
role (as previously shown for histones [52]). In addi-
tion, the molecular chaperone Hsp90 has been shown
to interact with RPS3 and RPS6, protecting them from
ubiquitination and proteasome-dependent degradation
[50]. Ubiquitination also has a role in ribosome biogen-
esis. In fact, it has been shown that proteasome inhibi-
tion alters both rRNA gene transcription and
maturation of the 90S preribosome complex; it also
leads to the depletion of 18S and 28S [51]. Moreover,
ubiquitin molecules on RPs can promote ribosome
assembly. In fact, in eukaryotes, RPL40, RPS27a and
RPP1 are synthesized as ubiquitin fusions, although the
ubiquitin part is then removed by post-translational
modification [53,54]. The transient association between
ubiquitin and RPs can promote their incorporation
into mature ribosomes, and is required for efficient
ribosome biogenesis. Another post-translational modifi-
cation of RPs has been shown by Hay et al., who, in
the search for novel proteins modified by neural-pre-
cursor-cell-expressed developmentally downregulated 8
(NEDD8) conjugation, identified 36 RPs from both
small and large subunits [55]. NEDD8 is a ubiquitin-
like molecule involved in the regulation of protein sta-
bility that can modulate cell proliferation and survival.
Its best characterized substrates are members of the
cullin family of proteins [56]. NEDDylation can have
opposite effects on the stability of its molecular targets:

it stimulates cullin degradation [57], but increases RP
stability. An additional mechanism of ribosome degra-
dation that involves autophagy has been characterized
in a recent report from the Peter laboratory [58]. Auto-
phagy is a highly conserved catabolic mechanism for
degrading proteins and organelles such as mitochondria
(mitophagy), peroxisomes (pexophagy), and endoplas-
mic reticulum (reticulophagy). Kraft et al. have identi-
fied, together with nonselective processes, a novel type
of selective autophagy that they term ‘ribophagy’, and
that occurs in S. cerevisiae upon nutrient starvation.
Ribophagy requires an intact autophagy machinery
[cells deficient in autophagy-related gene 7 (Atg7) fail
to degrade ribosomes] and the ubiquitin protease
Ubp3p together with its cofactor Bre5 [whose mamma-
lian homologs are ubiquitin-specific protease 10
(USP10) and G3BP respectively]. Ubiquitination plays
an important role in this kind of selective autophagy,
because ribosomes need to be ubiquitinated in the early
steps of ribophagy for the recognition of the autopha-
gic membranes. Subsequently, ubiquitin molecules have
to be removed for the completion of the autophagic
process. Interestingly, even if both ribosomal subunits
are degraded by ribophagy, only 60S requires the
ubiquitin protease complex ubiquitin-specific protease 3
(Ubp3p)–Bre5p. This ribosome-specific autophagic
mechanism could also be involved in regulating the
amount of ribosomes according to cellular growth con-
ditions, or could act as a quality control mechanism
able to remove damaged or wrongly assembled ribo-

somes (summarized in Fig. 1).
RPs in human pathologies and animal
models
Ribosome deficiencies due to mutations in the genes
coding for RPs or for rRNA have been known for
many years in Drosophila and Xenopus [59–61]. In both
cases, the main phenotype is slow growth, as expected
in the case of protein synthesis impairment. It was
quite surprising, therefore, that mutations were identi-
fied in the RPS19 gene as being the cause of Dia-
mond–Blackfan anemia (DBA) [62]. In fact, this
syndrome is characterized principally by defective
erythropoiesis associated with a variable degree of
growth retardation and malformations. Most RPS19
mutations are whole gene deletions, translocations, or
truncating mutations (nonsense or frameshift), suggest-
ing that haploinsufficiency is the basis of DBA patho-
logy. However, several missense mutations have also
been described [63]. The recent finding that mutations
in other RPs are also involved in DBA strongly sug-
gests that a ribosomal failure is responsible for the
clinical phenotype. Among DBA patients, mutations
have been found in RPS19 (25%), RPL5 (9%), RPL11
(6%), RPL35a (3%), RPS24 (2%), RPS17 (1%), and
RPS7 (< 1%) [62,64–67]. At present, these mutations
account for about 50% of DBA cases, and other
mutated RPs could therefore be found. Although an
additional tissue-specific role for the involved RPs
[68,69] cannot be ruled out, the most likely hypothesis
is that erythropoiesis is the human developmental pro-

cess that is most sensitive to ribosomal defects. Consis-
tent with this model, it has been recently shown that
5q- syndrome is caused by a defect in RPS14 [70]. The
hematological phenotype of this syndrome (macrocyto-
sis, erythroid hypoplasia, increased risk of leukemia) is
strikingly similar to DBA, thus confirming the impor-
tance of ribosome function in erythropoiesis. Further
support for this hypothesis can be obtained by the
analysis of other human pathologies, such as dyskera-
tosis, cartilage-hair hypoplasia, and Shwachman–
Diamond syndrome [71]. All three diseases depend on
alterations of some aspects of ribosome biogenesis
and, besides specific clinical phenotypes, they all share
defective hematopoiesis. Analysis of the molecular
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3204 FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS
mechanism of DBA in cultured cells showed that alter-
ation of any of the involved RPs can affect the matu-
ration of rRNA [72]. Moreover, investigations on the
effect of mutations on the synthesis of RPS19 showed
that: (a) mutations that affect mRNA structure cause
a decrease in RPS19 mRNA level [73,74]; and (b) mis-
sense mutations affect the stability of the protein more
or less severely according to the position within the
amino acid sequence [75,76]. An explanation for the
hematological phenotype of ribosome pathologies
could be that, because erythroid progenitor cells prolif-
erate extraordinarily rapidly and need to accumulate
high concentrations of globin proteins, they require a
high level of ribosome biogenesis. The failure to meet

such requirements would trigger apoptosis, possibly
through specific mechanisms (ribosomal stress). The
several animal models with RP deficiency reported in
the literature only partially support this hypothesis.
The first alteration of an RP in mice was an inducible
deletion of both copies of the RPS6 gene in the liver
of adult mice [77]. In this study, the altered response
to partial hepatectomy suggested the existence of a
novel checkpoint preventing cell cycle progression as a
consequence of a defect in ribosome biogenesis. Subse-
quently, the same research group showed that genetic
inactivation of p53 in RPS6-haploinsufficient mouse
embryos bypassed the observed blocking of the cell
cycle at gastrulation [embryonic day (E) 5.5]. The res-
cued embryos developed until E12.5, when they died
with diminished fetal liver erythropoiesis and placental
defects [78]. A less severe phenotype was observed in
the belly spot and tail mouse mutation, which is a
deletion in the RPL24 gene causing a splicing defect.
Belly spot and tail homozygotes die before E9.5, but
the heterozygotes reach adulthood, although they are
smaller than wild-type littermates [79]. More specific
phenotypes of Bst ⁄ + mice include alterations in pig-
mentation (white ventral midline spot, white hind feet),
skeletal abnormalities (kinked tail), and defects in reti-
nal development. An even less drastic phenotype is
observed in the case of mutations of the RPL29 gene.
In fact, mice lacking one of the two alleles develop
normally, and even RPL29-null animals are viable. A
delay in global growth is, however, observed in null

embryos around mid-gestation [80]. This results in pro-
portionally smaller organs and smaller stature. In addi-
tion, fibroblasts from RPL29-null embryos show
decreased rates of proliferation and protein synthesis.
Therefore, RPL29 is dispensable for embryonic develop-
ment, although ribosomes without this protein may
work with reduced efficiency. Alteration of RPL22 also
has a mild effect on the organism. Relative to control
littermates, RPL22
) ⁄ )
mice show no evident differ-
ences in growth rate and size [81]. RPL22 deficiency,
however, selectively arrested development of a specific
T-cell lineage by inducing cell death. It is noteworthy
that knockdown of p53 blocked cell death and restored
thymocyte development. This suggests that, in addi-
tion to RPS6, RPL22 deficiency can also activate a
p53-dependent checkpoint, albeit, in this case, only in
specific cell types. A role of p53 in mediating the effect
of RP deficiency was also shown in a recent publica-
tion by McGowan et al. [82]. In a chemical mutagenesis
screen in mice for pigmental abnormalities, missense
alterations of RPS19 and RPS20 were identified in two
mutants with dominantly inherited dark skin in ears,
footpads, and tail (Dsk3 and Dsk4). In addition,
Dsk3 ⁄ + mice showed a slightly reduced erythrocyte
level, increased apoptosis of erythroid precursors, and
reduced body weight. Pigmentation alteration could be
reproduced by conditional deletion of one copy of
RPS6 in keratinocytes. All phenotypes (pigmentation,

red cells, growth) are dependent on the increase in
p53. Hyperpigmentation is therefore due to stimulation
of the production of Kit ligand in keratinocytes, which
in turn causes melanocytosis. Another mouse knockout
model for RPS19 produced results partially in contrast
with this last report. In fact, the RPS19
) ⁄ )
animals die
prior to implantation, whereas heterozygous mice have
a normal phenotype, including the hematopoietic sys-
tem [83]. Finally, interesting new information has also
been obtained from zebrafish models. Amsterdam
et al. [84] reported that many RP genes may act as
tumor suppressors. Moreover, tumors due to RP hap-
loinsufficiency show defects in p53 synthesis, suggest-
ing that appropriate amounts of RPs are required for
p53 protein production in vivo, and that disruption of
this regulation could contribute to tumorigenesis [85].
In other studies, RP deficiency was induced by inject-
ing antisense oligonucleotide analogs (morpholinos)
into one-cell-stage zebrafish embryos. The reduced
amounts of RPS19 and several other RPs caused
hematopoietic and developmental abnormalities similar
to DBA [86,87]. Interestingly RPL11-deficient embryos
display abnormalities mostly in the brain [88]. Simi-
larly to some mouse models, RP deficiency in zebrafish
seems to activate a p53-dependent checkpoint that
induces developmental abnormalities [86,88]. The
affected tissues, however, could be different according
to the RP involved. Vertebrate animal models for RP

deficiency are summarized in Table 1.
Conclusions
In the last few years, the study of the synthesis and
function of RPs has both expanded our knowledge
S. Caldarola et al. Ribosomal protein synthesis
FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS 3205
and highlighted the issues that still need to be solved.
Translational regulation of RP synthesis associated
with the growth status of the cell has been known for
more than 20 years. The cis-acting sequences responsi-
ble for the regulation were identified in the early
1990s, but the trans-acting factors involved are still
unknown, and the few hypotheses proposed remain
unconvincing. Further disappointment came from
research that disproved the widespread model of the
role of S6Ks and phosphorylated RPS6 in TOP
mRNA translational regulation. Nevertheless, stimulat-
ing results were obtained from the analysis of RP turn-
over and investigations into the effects of RP
mutations in animal models and human pathologies. A
role for protein turnover in RP gene expression was
proposed in early studies on ribosome biogenesis [89].
However, the observation that RPs are produced in
excess and then rapidly degraded in the nucleolus [20]
is surprising. A rationalization of this apparent waste
of energy could be that the ribosomes are so important
that they justify a certain degree of redundancy in their
synthesis. This idea, however, conflicts with the finely
tuned regulation at the translational level observed in
response to growth factors, nutrient sufficiency, etc.

New studies on RP turnover have opened up a
scenario of additional regulatory mechanisms in RP
Table 1. Vertebrate animal models with RP alterations.
RP Organism Alteration Phenotype p53 inhibition References
RPS6 Mouse Conditional deletion (liver) Cell cycle block Not done [77]
RPS6 Mouse Deletion ) ⁄ +: embryonic lethal Partial rescue [78]
RPS19 Mouse Deletion ) ⁄ +: no phenotype
) ⁄ ): lethal
Not done [83]
RPS19 Zebrafish Knock-down Hematopoietic and developmental
abnormalities
Rescue [86,87]
RPS19, RPS20 Mouse Missense mutations
(Dsk3 and Dsk4)
Dsk ⁄ +: alteration of pigmentation,
erythrocyte development
Dsk ⁄ Dsk: lethal
Rescue [82]
RPL11 Zebrafish Knock-down Brain abnormalities, lethal Rescue [88]
RPL22 Mouse Deletion ) ⁄ +: no phenotype
) ⁄ ): viable, defect in alpha–beta
T-cells
Rescue [81]
RPL24 Mouse Missense mutation (Bst) Bst ⁄ +: alteration of pigmentation,
skeleton and retinal development
Bst ⁄ Bst: lethal
Not done [79]
RPL29 Mouse Deletion ) ⁄ +: no phenotype
) ⁄ ): viable, mild growth retardation
Not done [80]

Fig. 3. p53-dependent ribosomal stress.
Defects of ribosome biogenesis at any step
lead to the activation of p53 and conse-
quently to block of the cell cycle or apop-
tosis. Red crosses indicate steps that may
be affected.
Ribosomal protein synthesis S. Caldarola et al.
3206 FEBS Journal 276 (2009) 3199–3210 ª 2009 The Authors Journal compilation ª 2009 FEBS
synthesis involving small modifying peptides (ubiquitin
and NEDD8) affecting protein stability. The most
intriguing recent finding is the phenotype resulting
from mutations in RPs in zebrafish, mice, and humans.
The studies of mutations in various RPs in the different
organisms identified both a common effect and species-
specific and RP-specific alterations. The general
consequence of RP alteration is the activation of a p53-
dependent ‘ribosomal checkpoint’. This is the first
response of the cell to a ribosome defect, and consists of
blocking of the cell cycle and ⁄ or activation of apoptosis
mediated by an increase in p53 levels (Fig. 3). The pre-
valent effect downstream of this checkpoint appears to
be an alteration of hemopoiesis, especially in humans.
The same phenotype is also partially observed in mice;
however, here, an alteration of pigmentation seems to
prevail. Why erythroid differentiation and melanocyto-
sis are more sensitive to ribosome defects remains
unclear, and the difference between mice and humans is
puzzling. Similarly unexpected are the RP-specific effects
observed in mice. The explanation of a supplementary
role of a few RPs, although demonstrated in some cases,

is not entirely convincing. A more intriguing interpreta-
tion is a possible specific functional role of the various
RPs within the ribosome, as recently observed in yeast
[90]. As a consequence, RPs could be more or less
important for ribosome functioning, consistent with the
variable impact of mutations in different RPs observed
in mice (e.g. RPS6 > RPS19 > RPL22 > RPL29; see
also Table 1). A further extension of this hypothesis
could be heterogeneity in the composition of the ribo-
some, as shown in Ascaris [91], although there is no evi-
dence for this in vertebrates. Another possibility that
could partially explain the different impacts of muta-
tions in diverse RPs is a variable basal level (of both
mRNA and ⁄ or protein) in different tissues and ⁄ or
species. Despite some evidence for variability in the
amounts of RPs in different tissues, this aspect has not
yet been thoroughly analyzed. A final remark is that the
identification of human pathologies dependent on RP
mutations has stimulated interest in this group of basic
cell components. This has already helped to step up
research in this field, and will hopefully clarify issues
that remain unsolved.
Acknowledgements
We thank V. Iadevaia for the artwork. The financial
support of Telethon–Italy (Grant no. GGP07241A to
F. Loreni) is gratefully acknowledged. This work was
also supported by the Diamond Blackfan Anemia
Foundation, Inc. and the Italian Ministry of Univer-
sity and Research (FIRB and PRIN grants).
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