Synthesis and function of ribosomal proteins – fadingmodels and new perspectives Sara Caldarola, Maria Chiara De Stefano, Francesco Amaldi and Fabrizio Loreni Department of Biology, Univ
Trang 1Synthesis 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: loreni@uniroma2.it
(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.
Trang 2and 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 initiatransla-tion factors, translatransla-tion 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
a 5¢-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
Trang 3TOP 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.
Trang 4final 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
Trang 5from 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 regulainitia-tion 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).
Trang 6cell 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
Trang 7mechanism 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
Trang 8and 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.
) ⁄ ): lethal
abnormalities
RPS19, RPS20 Mouse Missense mutations
(Dsk3 and Dsk4)
Dsk ⁄ +: alteration of pigmentation, erythrocyte development Dsk ⁄ Dsk: lethal
) ⁄ ): viable, defect in alpha–beta T-cells
RPL24 Mouse Missense mutation (Bst) Bst ⁄ +: alteration of pigmentation,
skeleton and retinal development Bst ⁄ Bst: lethal
) ⁄ ): viable, mild growth retardation
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.
Trang 9synthesis 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 funcinterpreta-tional 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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