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. Author manuscript; available in PMC: 2010 Apr 10.
Published in final edited form as: Mol Cell. 2009 Apr 10;34(1):3–11. doi: 10.1016/j.molcel.2009.03.006

How Common are Extra-ribosomal Functions of Ribosomal Proteins?

Jonathan R Warner 1,*, Kerri B McIntosh 1
PMCID: PMC2679180  NIHMSID: NIHMS103450  PMID: 19362532

Abstract

Ribosomal proteins are ubiquitous, abundant, and RNA-binding, prime candidates for recruitment to extra-ribosomal functions. Indeed, they participate in balancing the synthesis of the RNA and protein components of the ribosome itself. An exciting new story is that ribosomal proteins are sentinels for the self-evaluation of cellular health. Perturbation of ribosome synthesis frees ribosomal proteins to interface with the p53 system, leading to cell cycle arrest or to apoptosis. Yet in only a few cases can we clearly identify the recruitment of ribosomal proteins for other extra-ribosomal functions. Is this due to a lack of imaginative evolution by cells and viruses, or to a lack of imaginative experiments by molecular biologists?

Ribosomal Proteins as candidates for Extra-ribosomal Functions

Ribosomal proteins (RPs) are abundant RNA-binding proteins found in every cell. It seems likely that they would be recruited to carry out many auxiliary functions, particularly by the viruses, which are so adept at usurping the cellular machinery. Indeed, a recent review claimed “Moonlighting is particularly widespread among ribosomal proteins, many of which have extra-ribosomal employment.” (Weisberg, 2008). The purpose of this review is to evaluate that statement. Some thirteen years ago Wool listed more than 30 potential extra-ribosomal functions of RPs (Wool, 1996), and a current review has pointed out some of the more recent ones (Lindstrom, 2009).

As a ribonucleoprotein particle responsible for the synthesis of new proteins in every cell of every organism, the ribosome has many features that are universal. These include the fundamental three dimensional structures of the RNA molecules of the small and the large ribosomal subunits, which carry out, respectively, the precise interaction of mRNA codon with tRNA anticodon and, some 75Å distant, the catalysis of peptide bond formation. The rRNAs are assisted in these functions, or at least in their folding to perform these functions, by a large number of evolutionarily conserved proteins, 50-54 for eubacteria, 57-68 for archaea, and 79-81 for eukaryotes (Lecompte et al., 2002). [L1,2,3… and S1,2,3… refer to proteins derived from the large and small subunits, respectively. Regrettably, the names do not always denote the same protein from different organisms. Useful glossaries are at http://ribosome.med.miyazaki-u.ac.jp/rpg.cgi?mode=genetable and http://mips.gsf.de/proj/yeast/reviews/rib_nomencl.html].

As RNA-binding proteins, most RPs are very basic, with a pI of >10. Exceptions are the acidic phosphoproteins, L7/L12 of prokaryotes and P0-P3 of eukaryotes, which form the stalk that interacts with translation factors. P0, as an auto-antigen often associated with neuro-psychiatric lupus erythematosus (Elkon et al., 1985; Kiss and Shoenfeld, 2007), demonstrates a rather unwelcome extra-ribosomal function.

Although not an extra-ribosomal function, it is worth noting that in organisms from yeast to man the genes for two RPs encode an extra-ribosomal protein: ubiquitin is N-terminally fused to L40 and S27a (Finley et al., 1989; Catic and Ploegh, 2005), but is immediately cleaved from the translation product. In rapidly growing S. cerevisiae this is the major source of ubiquitin.

What criteria are needed to conclude that a RP is acting in an extra-ribosomal capacity? We suggest three: (1) demonstration that the RP in question interacts specifically with some non-ribosomal component of the cell, presumably RNA or protein, (2) demonstration that such an interaction has a physiological effect on a living (or dying) cell, and (3) evidence that the latter is occurring away from the ribosome.

Although many protein-protein interaction studies and genetic screens have identified RPs or their genes, we will argue that based on these criteria the identifications, with a few significant exceptions, fall into one of three categories: (a) extra-ribosomal functions to control balance among ribosomal constituents, (b) extra-ribosomal functions that identify nucleolar stress, or aberrant ribosome synthesis, leading to cell cycle arrest or apoptosis, and (c) observations that implicate RPs but with little evidence of a specific extra-ribosomal function, observations that could be caused by specific or non-specific effects at the ribosome. [We have excluded reports of in vitro functions of RPs that have not been confirmed in vivo.]

Early Evidence of Extra-ribosomal Functions

The concept of extra-ribosomal functions of RPs was initiated with the observation in 1974 that bacteriophage Qβ encodes a polypeptide which joins with three E. coli host proteins, the translation factors EFTu and EFTs and the RP, S1, to serve as the RNA replicase responsible for replication of its genome (reviewed in (Blumenthal and Carmichael, 1979)). The function of the S1 component may be to bind to specific sites on the Qβ genome.

Studies of bacteriophage λ transcription identified a NUS (N utilization) complex necessary for certain transcription termination events during bacteriophage λ infection. One component of the NUS complex, the host NusE protein, is in fact S10 (Friedman et al., 1981). Recent structural work has shown that S10, together with NusB, another host protein, interacts with specific regions of λ transcripts, and can do so only when it is not associated with the ribosome (Luo et al., 2008). S10 has a globular portion that sits at the ribosome surface and an extended loop that penetrates into the 30S ribosomal subunit. The latter is essential for ribosome function but not for NUS activity. The NUS complex can effect either termination or anti-termination, depending on the context (reviewed in (Roberts et al., 2008)). Interestingly, the NUS complex functions as an anti-terminator for rRNA transcription. Thus the presence of S10 in the NUS complex provides one way in which rRNA and RPs can be coupled, i.e., a deficiency of S10 will lead to less anti-termination and less rRNA, and vice versa (reviewed in (Squires and Zaporojets, 2000)). Recent evidence suggests that S4 can act in a similar way (Torres et al., 2001). We will return to such coupling below.

Extra-ribosomal functions within the Ribosome System

For ribosome synthesis stoichiometry is essential, requiring equimolar production of rRNA and some 70 to 80 proteins (Perry, 2007). As shown by the Nomura, Lindahl, and other groups many years ago, this function is carried out in E. coli largely by the extra-ribosomal use of RPs as translational repressors of RP synthesis (reviewed in (Zengel and Lindahl, 1994; Nomura, 1999)). As an example, the S10 operon encodes eleven different RPs, one of which is L4. When bound to a specific structure in the 5′UTR of the S10 operon mRNA (Stelzl et al., 2003) L4 causes inhibition of translation of the entire operon. Thus, to a first approximation, each of the nineteen operons containing RP genes is constitutively transcribed. Each of these mRNAs has a binding site for a single RP. A newly translated protein can bind either to newly transcribed rRNA or to its own mRNA. If there is insufficient rRNA, the protein will bind to the mRNA to repress translation. In that way the production of RPs is balanced with the production of rRNA. Thus the regulation of ribosome synthesis, which uses a major fraction of the cell's energy resources, can occur primarily at a single step, the transcription of rRNA (Gaal et al., 1997). In many cases the structure of the mRNA to which the RP binds resembles the structure of the rRNA with which it associates in the ribosome. L4 is unusual in that it can also cause transcriptional attenuation of the S10 operon (Zengel and Lindahl, 1994). The cell's use of S10 as a participant in the NUS-mediated anti-termination complex mentioned above demonstrates that cross-talk occurs in both directions, with the clear evolutionary aim of balancing the production of rRNA and RPs.

L4 is quite remarkable. In addition to regulating the transcription and the translation of the S10 operon, more recent work identified a truly extra-ribosomal function: binding of L4 to RNAse E modulates its activity, leading to substantial changes in mRNA composition in response to stress (Singh et al., 2009).

In eukaryotic cells such auto-regulation of RP production is less pervasive, but there are several documented cases of RPs regulating their own synthesis. In S. cerevisiae L30 inhibits splicing by binding to its own transcript, thereby stabilizing a kink-turn structure that includes the 5′ splice site (Eng and Warner, 1991). S14 controls the splicing of the transcript of one of its genes (Fewell and Woolford, 1999). S28 binds to the 3′ UTR of its mRNA to stimulate its deadenylation and degradation (Badis et al., 2004). L2 appears to control the level of its mRNA through accelerated turnover (Presutti et al., 1991). In C. elegans, L12 and perhaps other RPs control their own synthesis by inhibiting the splicing of their own mRNAs (Mitrovich and Anderson, 2000). In human cells, S13 binds to the first intron of its transcript to inhibit splicing (Malygin et al., 2007). The convenience of a built-in RNA binding domain predicts that many such cases may be found.

In summary, evolution has employed the RNA binding characteristics of RPs to balance the synthesis of individual RPs with that of rRNA and with each other. This is clearly an extra-ribosomal function, but not in an extra-ribosomal system.

Surveillance of Ribosome Synthesis

Ribosome assembly in eukaryotic cells is exceedingly complex, requiring more than 200 protein and RNA molecules (reviewed in (Fatica and Tollervey, 2002)). The RPs participate in the assembly process. Lack of a RP can doom a nascent subunit, leading to potential accumulation of the other RPs of the subunit, with sometimes far-reaching effects, as we shall see.

Increasing evidence indicates that surveillance of ribosome assembly plays an important role in a cell's self-evaluation, in which defects in ribosome synthesis can lead to cell cycle arrest or apoptosis through extra-ribosomal functions of RPs. Thus far this seems to be an evolutionary adaptation largely confined to vertebrates.

5S rRNA assembles with L5 and L11 as a complex before being inserted into the large ribosomal subunit (Steitz et al., 1988; Zhang et al., 2007). Fifteen years ago, it was found in murine cells that L5 and 5S RNA could associate with MDM2 protein, as well as with MDM2-p53 complexes (Marechal et al., 1994). The identification of MDM2 (and its human orthologue HDM2) as an E3 ligase responsible for the ubiquitination of p53, leading to its rapid degradation (reviewed in (Vazquez et al., 2008)), suggested a functional role for the interaction of L5 with MDM2. Subsequently, it was shown that L11 could bind HDM2, confining it to the nucleolus, and that overexpression of L11 could lead cells to apoptose due to the accumulation of p53, whose E3 ligase was unavailable to initiate its destruction (Lohrum et al., 2003; Dai et al., 2004; Zhang et al., 2003). Interestingly, although L5 and L11 can individually bind MDM2, their activity in protecting p53 is amplified if both, as well as 5S rRNA, are present (Horn and Vousden, 2008). While these very basic proteins bind to an acidic domain of MDM2 (Lohrum et al., 2003), the importance of the Zn finger domain of the acidic region (Lindstrom et al., 2007), suggests a more specific type of interaction. L23 can also bind MDM2; overexpression of L23 causes cell cycle arrest or apoptosis. Intriguingly, siRNA-mediated suppression of L23 synthesis also leads to p53 accumulation and its downstream effects (Dai et al., 2004; Jin et al., 2004). Following on earlier hints that reducing synthesis of RPS3a in tumor cells (Naora et al., 1998) or increasing S29 (Khanna et al., 2000) would lead to apoptosis, it has now been shown that a constituent of the small ribosomal subunit, S7, can interact with the MDM2-p53 complex, again protecting p53 so that its effective concentration rises (Chen et al., 2007).

RP accumulation can also arise from imbalance between RPs and rRNA, e.g., in response to a low dose of actinomycin, which specifically reduces rRNA synthesis, to mycophenolic acid, which reduces the level of guanine nucleotides, and to 5-FU, which leads to inhibition of rRNA processing. Counterbalancing such accumulation are systems that bring about rapid turnover of unused RPs (Warner, 1977; Maicas et al., 1988). Nevertheless, in all three cases p53 accumulates, but knockdown of L5 or L11 impairs p53 accumulation (Lohrum et al., 2003; Sun et al., 2007; Sun et al., 2008).

p53 accumulation, and its consequent cell cycle arrest or apoptosis, occurs not only through imbalance among RPs but also through defects in a number of the many factors that are involved in the processing of precursor to mature ribosomes, e.g., Bop, (Pestov et al., 2001; Strezoska et al., 2002), nucleolin (Daniely et al., 2002), nucleophosmin (Colombo et al., 2002) and nucleostemin (Ma and Pederson, 2007). In the last case both L5 and L11 are necessary for the accumulation of p53 and the consequent G1 arrest (Dai et al., 2008).

The story becomes more baroque, however. In addition to p53, MDM2 can ubiquitinate L26, marking it for destruction (Ofir-Rosenfeld et al., 2008). Conversely, L26 can bind p53 mRNA, stimulating its translation (Takagi et al., 2005). Since L26 in the ribosome is located distal to the route of mRNA, it seems likely that here it is acting extra-ribosomally. L11 can bind the auto-ubiquitinated MDM2 to prevent its destruction by the proteasome (Dai et al., 2006). Finally, L11 can bind and sequester c-myc, itself a positive promoter of ribosome synthesis, and particularly of L11 synthesis, another example of the feedback systems evolved to regulate ribosome synthesis (Dai et al., 2007).

In some cases RPs appear to play a direct role in growth regulation. Nucleophosmin, a ribosome assembly factor, also acts a co-activator with the myc antagonist Miz1, a negative regulator of cell proliferation. L23 binds nucleophosmin, sequestering it in the nucleolus, thus reducing Miz1 activation and promoting cell proliferation (Wanzel et al., 2008).

Thus, we are gradually developing a picture of the complex choreography through which RPs serve an extra-ribosomal function as sentinels to warn of defects in ribosome assembly. Indeed it has been suggested that apoptosis is particularly responsive to signals of stress from the nucleolus, the primary result of which is disruption of ribosome synthesis (Rubbi and Milner, 2003). Accumulation of RPs can occur due to defects in ribosome assembly caused by an imbalance among RPs, caused by an imbalance between RPs and rRNA, or caused by a defect in one of the hundred or more proteins that catalyze the assembly process. Such an accumulation of any of several (most?) RPs can lead to accumulation of p53, either slowing p53 degradation by sequestering MDM2, or stimulating p53 translation.

There are some apparent contradictions between the various reports. For example, why should knockdown of L11 alone suppress p53 accumulation in response to inhibition of rRNA transcription (Bhat et al., 2004)? We would expect accumulation of L23, S7, or the others mentioned above to sequester MDM2.

Perhaps more troubling are some basic observations about the process of ribosome synthesis. A recent report suggests that mammalian cells make a substantial excess of RPs, which is subsequently degraded (Lam et al., 2007). In mammalian cells a large fraction, 25-50% of the 5S RNA, in complex with L5, is not associated with ribosomes, presumably in storage for ribosome assembly (Steitz et al., 1988). Why do such excess RPs, or the excess 5S RNPs, not sequester MDM2, leading to p53 accumulation? Perhaps there are barriers between the nucleoplasm and the nucleolus of which we are not yet aware.

Ribosomal Proteins in Development, Apoptosis, and Cancer

Haploinsufficiency of RPs in diploid organisms leads to phenotypes that are usually considered to arise from an insufficient supply of ribosomes. Minute mutations of Drosophila, characterized by slow development, short bristles, and impaired fertility, are found with unusual frequency, and at many loci. Since the first identification of a minute as encoding a RP (Kongsuwan et al., 1985) 64 minute loci have now been identified as RP genes (Marygold et al., 2007). RP haploinsufficiency in plants is similar, resulting in growth retardation, morphological abnormalities and reduced fertility, e.g. (Degenhardt and Bonham-Smith, 2008). For the most part one can explain the minute phenotype as resulting from insufficient ribosomes, but a hint of future complexities was the observation that S6 haploinsufficiency frequently leads to tumors of the hematopoietic system (Watson et al., 1992).

Remarkably, insertional inactivation of any of 11 zebrafish RP genes, representing both large and small subunits, leads to a high incidence of a specific, unusual tumor (Amsterdam et al., 2004). While normal cells accumulate p53 due to haploinsufficiency for a ribosomal protein, these tumors fail to do so. This is due not to effects on p53 transcription or on p53 turnover, but to specific inhibition of the translation of apparently normal p53 mRNA (MacInnes et al., 2008). We suggest that these tumors may arise by selection for a mutation that prevents this translation, as a mirror image of the demonstration in human cells that L26 can bind to the 5′UTR of p53 mRNA to promote its translation (Takagi et al., 2005).

Morpholino-induced knock-down of several RPs in zebrafish embryos, presumably more drastic than haploinsufficiency, extends the heterogeneity of response. Whereas knock-down of S19 leads to anemia, presumably due to apoptosis in the erythropoietic system (Uechi et al., 2008; Danilova et al., 2008), L11 knockdown leads more directly to head defects, coupled to an increase of p53 (Chakraborty et al., 2009). The overall message is that knock-down of many individual RPs leads to p53 accumulation, cell death, and defective development (Uechi et al., 2006; Danilova et al., 2008).

Mutations in either the S19 or S20 genes can lead to developmental defects in mice, including abnormal melanocyte proliferation and red blood cell hypoplasia (McGowan et al., 2008). Although engineered knock-outs (KO) of relatively few mouse RP genes have been reported, most of these are embryonic lethal even as heterozygotes, e.g., S6 (Panic et al., 2006). (See http://www.informatics.jax.org/searches/allele_form.shtml for more details.)

A decade ago the identification of mutations in RP S19 as the cause of 25% of the cases of Diamond-Blackfan anemia (DBA) (Draptchinskaia et al., 1999) was not only a breakthrough in understanding that debilitating disease but also a wake-up call that the RPs, hitherto ignored as ‘housekeeping proteins’, could play a rather more complex role than previously imagined. Since then, several other RPs have been implicated in DBA, namely S24, S17, L35a, L5, and L11, and in a number of cases additional developmental defects as well as increased risk of cancer have been partially correlated with mutations of individual RP genes (Gazda et al., 2008).

Other cases of insufficient RPs leading to developmental defects have been reported. Deletion of a portion of chromosome 5, usually in older individuals, leads to severe anemia and to a propensity to progress to acute myeloid leukemia. This 5q-syndrome is due to the loss of the gene for S14 (Ebert et al., 2008). Human S4 is the only RP encoded by two genes, on the X and Y chromosomes. Turner's syndrome, characterized by short stature, degeneration of the gonads, and frequent intrauterine lethality, has been identified with insufficiency of S4 in females due to the failure to exempt the S4 gene from X inactivation (Fisher et al., 1990).

It is now somewhat easier to understand why haploinsufficiency for a RP can lead to such complex phenotypes during development, ranging from lethality to stunted growth to anemia to altered cell development and migration to tumorigenesis, or, indeed, to no apparent effects. These variable outcomes arise from the dual effects of haploinsufficiency: (a) insufficient ribosomes lead not only to inefficient translation but also to an altered spectrum of translation products because mRNAs are competing for scarce ribosomes, and (b) cell cycle arrest or apoptosis is brought about by the RP sentinels, at least in vertebrates. Both are likely to vary substantially among developing tissues, and even within tissues, depending on growth rates and specific proteins synthesized. The end stages of erythropoiesis, in particular, call for extremely rapid growth, division, and translation, which might make such cells uniquely susceptible to apoptotic signals.

Indeed, DBA patients show increased apoptosis in erythroid cells of the bone marrow (Rossi et al., 2007), and p53-mediated apoptosis is the cause of the developmental phenotypes observed in mice with mutations in S19 and S20 (McGowan et al., 2008) A vivid example is the mouse liver where both S6 genes have been experimentally ablated. Whereas partial hepatectomy normally leads to rapid cell growth and proliferation, in the absence of S6 some growth occurs, but no ribosomes are made, and absolutely no cell division occurs, as predicted if p53 is allowed to accumulate (Volarevic et al., 2000).

Finally, among cells whose nucleolar stress leads to p53 accumulation, with its resultant cell cycle arrest and apoptosis, there should be a strong selective advantage for those cells that have lost p53, or one of the components of its apoptotic pathway. Since this is a key step in tumorigenesis, it is not surprising that many instances of RP haploinsufficiency display an increased likelihood of cancer.

Authentic Extra-ribosomal functions

There are, indeed, a few cases where we can be confident that a RP is performing extra-ribosomal functions.

L7

In archaea L7 is part of the CD snoRNA complex involved in rRNA processing but has evolved in eukaryotes into the very similar 15.5 protein that performs the same function (Kuhn et al., 2002). This is a nice demonstration of an RP being recruited for an extra-ribosomal function and then, presumably after gene duplication, evolving into a non-ribosomal protein carrying out that function.

RACK1

Although the list of RPs was stable for many years, a newcomer has recently been added to the eukaryotic ribosome, thanks largely to the improved methods of mass spectrometry (Link et al., 1999). This is the protein termed RACK1 (‘receptor of activated C kinase’) which had previously been implicated as a scaffold in many signal transduction functions, e.g. (Kadrmas et al., 2007). The high codon index of the S. cerevisiae orthologue (ASC1), its intron, its transcriptional regulation by Fhl1 and Ifh1 (Kleinschmidt et al., 2006), and particularly its localization to the head of the small ribosomal subunit by cryo-EM (Sengupta et al., 2004) support the classification of RACK1 as a RP. On the other hand, unlike most RPs, RACK1 is not a permanent member of the ribosome; in stationary phase cells nearly half is not associated with ribosomes (Baum et al., 2004), and some of its structural/biochemical properties are inconsistent with it being fully ribosome-bound (Coyle et al., 2009). Thus RACK1 may serve not only as a powerful interface between the cell's signaling and translational machineries but also as a signal transduction agent far from the ribosome. The breadth of these extra-ribosomal functions make RACK1 unique among the RPs. (A current review is needed. Many authors seem unaware that RACK1 is primarily a RP.)

L13a

One very clear case of a RP being recruited for an extra-ribosomal function is L13a. Treatment of U937 cells with IFNγ leads to a kinase cascade, where death-associated protein kinase-1 (DAPK) activates zipper-interacting protein kinase (ZIPK), leading to phosphorylation of L13a at residue S77. As a result, L13a leaves the ribosome and joins a complex termed GAIT, consisting of glutamyl-prolyl tRNA synthetase, NS1-associated protein-1, and glyceraldehyde-3-phosphate dehydrogenase (Mazumder et al., 2003; Mukhopadhyay et al., 2008). Although the ribosomes are almost bereft of L13a, they continue to translate mRNA, and the cell continues to grow at a normal rate. The function of the GAIT complex is to inhibit the translation of specific mRNAs by binding a structure in their 3′UTR. Surprisingly, it is the tRNA synthetase that binds the mRNA, while the job of L13a is to interact with eIF4G to reduce the translation initiation complex (Kapasi et al., 2007). The target mRNA originally identified was that encoding ceruloplasmin, a multifunctional copper protein, but it now seems clear that there are many, including VEGF-A (Ray and Fox, 2007) as well as the DAPK and ZIPK kinase mRNAs, thus providing a nice example of negative feedback (Mukhopadhyay et al., 2008). Crystallographic analysis shows the archaeal L13a orthologue on the exterior of the ribosome; an interesting suggestion is that the ribosome, and other multi-component assemblies, could serve as a depot to store such regulatory proteins (Ray et al., 2007).

S3

S3 from Drosophila or mammalian cells can nick DNA at abasic sites (Wilson et al., 1994; Kim et al., 1995). Recent evidence shows that genomic damage can lead to a dose-dependent transfer of S3 from the cytoplasm to the nucleus as a result of ERK-mediated phosphorylation of S3 on T42, (Yadavilli et al., 2007), although a recent report suggests that PKCδ may do so as well (Kim et al., 2009). The physiological role of S3 is puzzling and may be context-dependent. For example, S3 appears to inhibit base excision repair of 8-oxoG residues: knockdown of S3 leads to substantial increase in survival of cells treated with H2O2 (Hegde et al., 2007). On the other hand S3 substantially increases the activity of uracil-DNA glycosylase, an initiating step in base excision repair (Ko et al., 2008). Remarkably, S3 can also enter the nucleus in response to TNF stimulation, where it becomes a part of the NF-κB complex, serving to stabilize the interaction of this transcription factor with specific sites in the genome (Wan et al., 2007).

L10

The Arabidopsis trans-membrane receptor kinase NIK1 can phosphorylate L10, triggering its translocation to the nucleus (Carvalho et al., 2008). This appears to be an anti-viral defence since loss of L10 results in the same susceptibility to geminivirus infection as seen in nik1 null mutants. As yet, the mechanism of action and the role of L10 in the nucleus is unclear, involving neither RNA silencing nor the hypersensitive response that characterize most known plant defense signaling cascades. Human L10, also known as QM, is an apparent jun-binding/inhibitory protein that can be translocated to the nucleus after interaction (phosphorylation?) with Pre-senilin-1 protein (Imafuku et al., 1999).

Hints of more to come, or red herrings?

There are an increasing number of observations that implicate RP in extra-ribosomal functions, but which lack mechanistic insight. We will mention only a few. An early genetic screen in S. cerevisiae found that overexpression of S20 would suppress a temperature sensitive mutant of RNA Pol III (Hermann-Le et al., 1994). No specificity has since been revealed. Yet, as this review was being submitted a new report suggests that L6 associates with Pol III transcribed genes and that its over-expression can suppress mutations in a Pol III specific transcription factor (Dieci et al., 2009), thus providing a potential coupling between different parts of the translation apparatus.

L22, and numerous other RPs, were found associated with histone H1 in Drosophila chromatin. Either overexpression or depletion of L22 had substantial effects on gene expression (Ni et al., 2006). What is the mechanism and specificity? L22 also binds strongly to EBER-1 RNA in Epstein-Barr virus-infected lymphocytes, but no function has yet been identified (Fok et al., 2006). A recent genome-wide association study has implicated S26, based on its reduced expression level, as a susceptibility gene for a form of type 1 diabetes (Schadt et al., 2008). If so, is S26 acting in a ribosomal or an extra-ribosomal capacity?

Finally, genome-wide transcriptome analyses suggest that the expression of specific RP genes is often elevated in different types of tumors (summarized in (Wang et al., 2009)). While there is no evidence that any of the encoded proteins are acting in an extra-ribosomal capacity, it is an intriguing possibility.

The Puzzling Observations in S. cerevisiae

A rich lode of potential extra-ribosomal functions has emerged from genome-wide screens in S. cerevisiae. Due to an ancient genome duplication event (Wolfe and Shields, 1997), most RPs of S. cerevisiae have two genes that encode identical or very similar amino acid sequences. In most cases both genes are active; either alone can support growth at nearly normal to somewhat slower rates (Dean et al., 2008). Genome-wide screens have identified deletions of specific RP genes as influencing a number of situations, such as bud site selection (Ni and Snyder, 2001), growth of diploid cells haploinsufficient for actin (Haarer et al., 2007), or life span (Steffen et al., 2008). Indeed, word-of-mouth reports suggest that nearly any screen using the yeast KO collection will yield hits with a number of RP genes. The intriguing feature of most such reports is that usually only one of the two paralogous genes shows such a phenotype (reviewed in (Komili and Roth, 2007)). Time will tell whether these intriguing results are due to extra-ribosomal functions.

It is worth pointing out that, unlike mammals, plants have several genes for most RPs, many of which are developmentally regulated (reviewed in (McIntosh and Bonham-Smith, 2006)). This situation provides a fertile opportunity for the evolution of extra-ribosomal functions.

Speculations

The criteria that we have imposed to establish extra-ribosomal function are difficult to achieve. As an example, suppose that HIV recruits a RP to bind to a 5′ splice site to regulate its splicing efficiency. How could that be identified? Owing to the abundance of RPs the virus need not use enough to affect the ribosomes or their synthesis. To deplete a cell of a RP experimentally is likely to lead to substantial changes in translation, thus masking, or at least muddling, identification of any extra-ribosomal effect of the protein. Attempts to purify the critical spliceosome would likely be frustrated by contamination with ribosomes. Indeed, the TAP tag analyses of yeast proteins usually discount RPs, as they are found in so many preparations (Gavin et al., 2002). In short, such an extra-ribosomal function could easily go undetected.

In summary, it is clear that ribosomal proteins can act off the ribosome to control the synthesis of ribosomal components. It is clear that ribosomal proteins can act off the ribosome to alert the cell to stress or defects in ribosome synthesis itself. Yet, at the end we can only express our surprise at the very few verified cases of ribosomal proteins being recruited for a function unrelated to the ribosome or its synthesis, our curiosity about the variety of phenotypes caused by aberrant (or insufficient) ribosomal proteins, and our expectation that in the end both cells and viruses will have proved themselves clever enough to develop many uses for the ubiquitous ribosomal proteins, uses they will keep to themselves until we are clever enough to uncover them.

Table 1.

Ribosomal Proteins with Extra-ribosomal Functions

Ribosomal Protein Extra-ribosomal Function References (* reviews)
Bacteriophage recruits
S1 Qβ replicase *(Blumenthal and Carmichael, 1979)
S10, S4 NUS complex ; Antitermination *(Roberts et al., 2008)
Autoregulation of RP synthesis (prokaryotes)
L4 (and many others) Inhibits translation of polycistronic mRNA *(Nomura, 1999)
L4 Inhibits transcription of S10 operon *(Zengel and Lindahl, 1994)
L4 Interacts with and influences RNAse E (Singh et al., 2009)
Autoregulation of RP synthesis (eukaryotes)
L30 (S.c.) Inhibits its own mRNA splicing (Eng and Warner, 1991)
S14 (S.c.) Inhibits its own mRNA splicing (Fewell and Woolford, 1999)
L2 (S.c.) Shortens its own mRNA T1/2 (Presutti et al., 1991)
S28 (S.c.) Shortens its own mRNA T1/2 (Badis et al., 2004)
L12 (and others?) (C.e.) Inhibits its own mRNA splicing (Mitrovich and Anderson, 2000)
S13 (H.s.) Inhibits its own mRNA splicing (Malygin et al., 2007)
Ribosome biosynthesis sentinels (mammals)
L5 Sequesters M/HDM2 Several—See Text
L11 Sequesters M/HDM2 Several—See Text
L23 Sequesters M/HDM2 Several—See Text
S7 Sequesters M/HDM2 (Chen et al., 2007)
L11 Sequesters c-myc (Dai et al., 2007)
L26 Promotes p53 translation (Takagi et al., 2005)
L23 Sequesters nucleophosmin from Miz1 (Wanzel et al., 2008)
Fully Extraribosomal
L7 (archaea) SnoRNP (Kuhn et al., 2002)
RACK1 Cell signaling (Link et al., 1999)
L13a Inhibits mRNA translation (GAIT complex) (Mazumder et al., 2003)
S3 DNA endonuclease (Wilson et al., 1994)
S3 Binds NFκB (Wan et al., 2007)
L10 (A.th) Antiviral (Carvalho et al., 2008)
L10 (H.s.) Binds c-jun (Imafuku et al., 1999)
Interesting possibilities
S20 (S.c.) Influences Pol III transcription (Hermann-Le et al., 1994)
L6 (S.c.) Influences Pol III transcription (Dieci et al., 2009)
L22 (and others) (D.m.) Binds Histone H1 (affects transcription?) (Ni et al., 2006)
L22 (H.s.) Binds EBER-1 RNA (of EB virus) (Fok et al., 2006)
S26 (H.s.) Susceptibility to Diabetes (?) (Schadt et al., 2008)
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This table lists ribosomal proteins that meet, at least partially, our criteria for performing extra-ribosomal functions. The literature describing the interaction of L5, L11, and L23 with M/HDM2 is complex, and the reader is referred to the text. ‘Interesting possibilities’ have not met at least one of the criteria. We have not included the large number of ribosomal proteins whose haploinsufficiency leads to altered phenotypes, as it is generally not known if such effects are due to extra-ribosomal functions.

Acknowledgments

We regret the omission of many important references due to space constraints. We are grateful for editorial comments from Lasse Lindahl, Ian Willis, and Michael Keogh. The research from this laboratory is supported in part by grants from the NIH: GM-25532 to JRW and CAI-3330 to the Albert Einstein Cancer Center. KBM is the recipient of a Postdoctoral Fellowship from NSERC of Canada.

Footnotes

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