Control points in eucaryotic ribosome biogenesis.

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Control points in eucaryotic ribosome biogenesis D. E. LARSON Department of Molecular Biology and Genetics, College of Biological Science, University of Guelph, Guelph, Ont., Canada NI G 2 W1

P. ZAHRADKA

Biochem. Cell Biol. 1991.69:5-22. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/03/15. For personal use only.

Department of Chemistry and Biochemistry, College of Physical and Engineering Science, University of Guelph, Guelph, Ont., Canada NIG 2 WI AND

B. H.

SELLS'

Department of Molecular Biology and Genetics, College of Biological Science, University of Guelph, Guelph, Ont., Canada N1G 2 W1 Received May 11, 1990

LARSON, D. E.,

ZAHRADKA,P., and SELLS,B. H. 1991. Control points in eucaryotic ribosome biogenesis. Biochem. Cell Biol. 69: 5-22. Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8s) and RNA polymerase 111 (SS), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase 11. Assembly of the ribosomal subunits in the nucleolus, the site of 45s rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems. Key words: rRNA, ribosomal proteins, nucleolus, ribosome.

LARSON, D. E., ZAHRADKA, P., et SELLS,B. H. 1991. Control points in eucaryotic ribosome biogenesis. Biochem. Cell Biol. 69 : 5-22. La biogtnkse des ribosomes dans les cellules eucaryotes implique la synthkse coordonnke de quatre espkces de rRNA, transcrits par la RNA polymerase I (18S, 28S, 5.8s) et la RNA polymCrase 111(SS), et environ 80 protkines ribosomiques traduites de mRNA synthktists par la rRNA polymCrase 11. L'assemblage des sous-unites ribosomiques dans le nucleole, le site de transcription du gkne prkcurseur du rRNA 45S, requiert le mouvement du rRNA 5S et des protkines ribosomiques qui passent respectivement du nuclCoplasme et du cytoplasme a cette structure. Pour integrer ces CvCnements et assumer la production kquilibrk des constituants ribosomiques individuels, les organismes eucaryotes ont dkveloppt diffkrentes strategies en reponse a divers changements physiologiques. Cette revue prCsente un resume des mtcanismes modulant la production des molecules prtcurseurs ribosomiques et le taux de biogkntse des ribosomes dans divers systkmes biologiques. Mots elks : rRNA, protkines ribosomiques, nucleole, ribosome. [Traduit par la Redaction]

Regulation of ribosome biogenesis One of the earliest events to occur following stimulation of cell growth is a change in the rate of ribosome precursor synthesis, suggesting a close relationship between the rates of cell proliferation and ribosome biogenesis (Maaloe and ETS, external transcribed spacer; ITS, internal ABBREVIATIONS: transcribed spacer; NTS, nontranscribed spacer; kb, kilobase@); bp, base pair(s); TIE-1, -IA, and -IB, transcription initiation factors 1, IA, and IB; hUBF, human upstream binding factor; xUBF, Xenopus upstream binding factor; TFIA, transcription factor IA; TTFI, transcription termination factor I; r-protein, ribosomal protein; snRNP, small nuclear ribonucleoprotein; snRNA, small nuclear ribonucleic acid; UAS, upstream activation sequences; RPG, ribosomal protein gene; kDa, kilodalton(s); TUF, TCMl yeast ribosomal protein gene upstream factor; TAF, translation activating factor; SUE, S33 yeast ribosomal protein gene upstream factor; NOR, nucleolar organizer region; ABAE, adult bovine aortic endothelial; bFGF, basic fibroblast growth factor; hsp, heat shock protein; CHO, Chinese hamster ovary. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprim6 au Canada

Kjeldgaard 1966; Nomura et al. 1984). The pioneering investigations of Maaloe and his colleagues with bacterial cells provided the basis for many of the studies which followed in eucaryotic systems. Perturbation of cell growth rate, whether increased by enrichment of the growth medium or decreased as in terminal differentiation, changes the rate of ribosome production in eucaryotes. For example, ribosome production can be arrested or reduced following cessation of growth in serum-starved (Emerson 1971; Cooper 1973) and stationary cells (Becker et al. 1971; Tushinski and Warner 1982), or following terminal differentiation (Bowman and Emerson 1977; Krauter et al. 1979; Jacobs et al. 1985; Bowman 1987a). Conversely, ribosome production can be increased by serum stimulation (Mauck and Green 1973), by hormone treatment (DePhilip et el. 1980), and during liver regeneration (Dabeva and Dudov 1982). In eucaryotic cells, ribosome biogenesis requires the equimolar accumulation of its various components. Synthesis of 18S, 28S, and 5.8s rRNAs is coupled, since these RNA species are processed from a single 45s rRNA primary


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transcript. In contrast, the fourth RNA component of the ribosome, 5s rRNA, is synthesized from a separate transcription unit. Balanced production of ribosomal proteins requires an even more complicated regulatory mechanism, since these genes are distributed among several chromosomes, necessitating a coordination between gene transcription and mRNA translation. The individual components are then assembled into large and small ribosomal subunits in the nucleolus and subsequently transported to the cytoplasm. Organisms have adopted diverse strategies to regulate production of ribosomal precursor molecules in response to ( i ) variations in nutrient levels, (ii) the presence of growth factors and hormones, and (iii) different stages of development. To determine which mechanisms modulate formation of the various components, much attention has been focused on elucidating the role of gene promoters and associated upstream DNA sequences, as well as trans-acting factors that work in concert with specific RNA polymerases, in controlling transcription of the different precursor genes. The study of ribosome production is further complicated by the fact that three different RNA polymerases are required. Whether communication and (or) interaction occurs amongst these enzymes has not been established. In addition, it has not been determined whether the level of ribosomal precursor components are controlled at the posttranscriptional level and whether specific nucleolar components play a role in regulating ribosome biogenesis. The 45s rRNA precursor Gene organization The 45s rRNA precursor gene is organized as a series of tandem repeating units containing clusters of hundreds to thousands of individual genes (Wellauer and Dawid 1979). Each mammalian transcription unit, in the 5 ' to 3 ' direction, consists of an ETS, 18s RNA, ITS containing 5.8s RNA, 28s RNA, and a NTS (reviewed by Mandal 1984). Synthesis of the primary transcript terminates in the latter region and is subsequently processed to give rise to mature 18S, 5.8S, and 28s rRNAs. In other systems, the general pattern of the 45s rRNA precursor transcription unit is similar, although large variations occur in transcript size, ranging from 8 kb in yeast, Drosophila, and Xenopus to 13 kb in mammals. Differences in the length of the ETS, ITS, and NTS regions are responsible for these size variations. Of note, the Drosophila 28s rRNA segment contains an intervening sequence and this may be common to all insect species. The entire transcription unit, because of these variations in spacer lengths, extends from 10 to 40 kb. Two cis-acting regions, located at - 40 to + 15 (core or proximal promoter) and - 150 to - 110 (upstream or distal promoter), regulate the expression of 45s rRNA precursor genes in all systems examined, including amoeba, rat, mouse, frog, and human cells (reviewed by Sollner-Webb and Tower 1986). The core promoter is required for minimum and efficient transcriptional initiation, while the upstream promoter is involved in modulating the level of synthesis. Both promoter elements have key roles in directing the binding of RNA polymerase I to the transcription initiation site. In addition, a specific sequence, located at - 171, prevents entry of RNA polymerase I from adjacent upstream 45s rRNA precursor genes (Bartsch et al. 1988). Since this element contains a sequence specifically recognized by the restriction endonuclease SalI, it has been designated

BIOL. VOL. 69, 1991 a Sal box, which also functions as a transcription terminator. From a point mutational analysis of this region, Firek et al. (1989) demonstrated that it can both augment and repress transcription. The nontranscribed spacer between adjacent 45s rRNA precursor genes consists of two to six repeats of a 1-kb region that augments rRNA gene transcription (reviewed by Sollner-Webb and Tower 1986; Labhart and Reeder 1989). Each region contains a duplicate rRNA gene promoter and 60/81-bp repetitive enhancer elements, which may function by binding to a RNA polymerase I transcription factor, which in turn augments binding to an adjacent promoter (Sollner-Webb and Tower 1986; Cassidy et al. 1986). These elements increase transcription independent of orientation, but function only when located upstream of an active rRNA gene. The ability of the promoter present in each 1-kb region to initiate RNA transcription (Kuhn and Grummt 1987) may be related to efficient rRNA gene expression, although how this may be accomplished remains controversial (Mitchelson and Moss 1987; Labhart and Reeder 1989). The presence of a terminator, such as T3 in Xenopus (Labhart and Reeder 1986), upstream of the gene promoter prevents continued transcription. In Xenopus, the NTS region between adjacent 45s rRNA precursor genes has additional regulatory sequences consisting of a 1-kb segment, present in multiple copies, which contains a duplicate promoter and 60/81-bp repetitive enhancer elements (Moss 1983; Labhart and Reeder 1989). These repeat regions have a stimulatory effect on gene transcription that can be exerted over large distances. Although it is not known how the 60/81-bp enhancer elements act to augment transcription, Sollner-Webb and Tower (1986) have suggested that the enhancer elements may act by binding to a polymerase I transcription factor, which in turn augments binding to an adjacent promoter. In rat rRNA genes, a trans-acting factor binds to the same region of the nontranscribed spacer region that exhibited enhancer activity (Cassidy et al. 1986). Trans-actingfactors involved in 45s rRNA gene transcription Initiation and termination of 45s rRNA precursor gene transcription are controlled by ancillary factors distinguished by their functional characteristics. These factors have been identified in cell extracts fractionated on a variety of column matrices and assayed in cell-free transcription systems. In Acanthamoeba, only two factors are apparently required for gene transcription. TIF-1 binds to the proximal promoter and directs RNA polymerase I to the transcription initiation site (Kownin et al. 1987), while a phosphorylated form of RNA polymerase I is thought to be essential for formation of the transcription initiation complex (Bateman and Paule 1986). The factors identified in vertebrate systems possess analogous functions to those found in Acanthamoeba. Two DNA-binding proteins, designated SL1 and hUBF, which have been isolated from human cells, cooperate to form the transcription initiation complex and recruit RNA polymerase I (Bell et al. 19886). hUBF, recently cloned by Tjian and his colleagues (Jantzen et al. 1990), binds to the downstream sequences ( - 75 to - 115) of the distal promoter and directs the binding of SL1 to both distal and core promoter regions, presumably through protein-protein interactions since SL1 alone does not bind to these sites. SLl is also responsible for the species-specific enhancement of


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transcription in human cells (Bell et al. 1989). In addition, the Xenopus factor xUBF, similar to hUBF, modulates rRNA gene transcription also by binding to enhancer regions (Pikaard et al. 1989). Chromatographic separation of cultured mouse cell extracts has led to the identification of a fraction containing proteins, independently designated factor D or TIF-IB, involved in recruiting RNA polymerase I to the transcription initiation complex (Mishima et al. 1982; Clos et al. 1986; Tower et al. 1986). Purification and characterization of TIF-IB has demonstrated that this factor has properties similar to SL1 (Schnapp et al. 1990). Another factor, which does not bind directly to DNA, may also be required for recruitment of RNA polymerase I into the transcription initiation complex. The nature of this component, designated C in mouse extracts, remains controversial. Although two interconvertible forms of RNA polymerase I differing in their chromatin-binding characteristics have been described, separate functions for each component have not been defined (Lampert and Feigelson 1974; Matsui et al. 1976; Kellas et al. 1977). Physical differences between the active and inactive forms of RNA polymerase I, noted during column chromatography and sedimentation analysis (Buttgereit et al. 1985; Cavanaugh et al. 1984; Tower and Sollner-Webb 1987), suggest factor C may represent a protein component closely associated with a transcriptionally active form of RNA polymerase I (Buttgereit et al. 1985; Cavanaugh and Thompson 1985). Alternatively, it may represent a modified subform of RNA polymerase I activated by phosphorylation, as in Acanthamoeba, which can stimulate the rate of RNA synthesis (Duceman et al. 1981). Since the activated RNA polymerase I constitutes only a small percentage of the total cellular content of the enzyme, it is technically difficult to distinguish between these two possibilities. Additional factors that may influence rRNA transcription in vivo have been identified by fractionation of cell extracts and monitoring transcriptional activity in vitro. For example, poly(ADP-ribose) polymerase reduces the formation of nonspecific transcripts in vitro (Kurl and Jacob 1985). Kato et al. (1986) identified TFIA, a factor involved in transcription initiation complex formation, which may facilitate multiple rounds of synthesis. Another factor, also designated TFIA, was shown to function as a ribonuclease inhibitor (Tower et al. 1986). The relationship between these two activities, both eluting in the same fraction from phosphocellulose, has not been established. A high mobility group like, DNA-binding factor that interacts specifically with the 5 ' upstream sequences of the rat rRNA gene also stimulates rRNA synthesis in vitro (Yang-Yen and Rothblum 1988). Whether these factors have a role in regulating rRNA transcription in vivo is currently unclear. Other factors, whose function cannot be detected by the cell-free assays typically used, may also be involved. A prime example is DNA topoisomerase I, which controls the changes in superhelical density of DNA during the transcription process (Futcher 1988). While this enzyme appears nonessential for gene transcription by RNA polymerase 11, its presence is necessary for efficient transcription of the 45s pre-rRNA gene in both yeast and mammalian cells (Brill et al. 1987; Rose et al. 1988; Zhang et al. 1988). In addition, a role for topoisomerases in both the structural organization of rRNA genes (Culotta and Sollner-Webb 1988) and formation of nucleoli (Hirano et al. 1989) has been suggested.

Nashimoto and Mishima (1988) have proposed a model for the regulation of rRNA transcription in which the existence of a transition pathway from the terminator to the promoter increases the rate of synthesis. The coordination between initiation and termination requires the involvement of two termination factors and provides a mechanism for rapid changes in rRNA transcription. Support for this model has been provided by characterization of TTFI, a protein which binds to the Sal box (Bartsch et al. 1988) and prevents read through of RNA polymerase I into the upstream promoter region via a specific interaction with the polymerase molecule (Bateman and Paule 1988; Henderson et al. 1989; Kuhn et al. 1990). In the absence of TTFI, factor D would be displaced from rDNA and unable to recruit RNA polymerase I molecules into new transcription initiation complexes. Modulation of rRNA synthesis Many of the early studies investigating cell growth demonstrated that ribosome production varies directly with the rate of cell proliferation. Regulation of 18S, 28S, and 5.8s rRNA synthesis appears to be a limiting step in the control of ribosome formation. Although rRNA and r-protein production are coordinately regulated in proliferating myoblasts, only rRNA synthesis is decreased in terminally differentiated myotubes which have a reduced requirement for ribosomes (Krauter et al. 1979, 1980; Jacobs et al. 1985). Similar observations documented in other systems have demonstrated that control of rRNA synthesis involves modulation of either the RNA polymerase I associated factor C (Buttgereit et al. 1985; Cavanaugh and Thompson 1985; Mahajan and Thompson 1987; Rubinstein and Dasgupta 1989) or the extent of RNA polymerase I phosphorylation (Bateman and Paule 1986; Tower and Sollner-Webb 1987). In general, the decrease in rRNA transcription is independent of the total cellular content of RNA polymerase I in the various systems studied. An exception to this trend has been noted during growth stimulation of cardiac myocytes, which display an increase in both rRNA synthesis and total RNA polymerase I activity (McDermott et al. 1989). A variety of physiological changes alter the rate of rRNA synthesis. Decreased rRNA gene transcription occurs during differentiation of MM14 myoblasts (Bowman 1987a) and HL-60 human leukemia cells (Schwartz and Nilson 1988). In HeLa cells and tomato cell cultures, rRNA synthesis is significantly reduced during heat shock (Nover et al. 1986; Parker and Bond 1989). The inhibition of rRNA synthesis following heat shock in yeast occurs in concert with a drop in r-protein gene transcription (Kim and Warner 1983; Veinot-Drebot et al. 1989). In contrast, rRNA gene activity is increased by insulin in MM14 myoblasts (Hammond and Bowman 1988), in regenerating rat liver (Dabeva and Dudov 1982), and in rat liver in response to glucocorticoid treatment (Frey and Seifart 1982; Dabeva and Ikonomova 1982). Changes in the rate of rRNA gene transcription have also been reported in plants (Tobin and Silverthorne 1985). Further experiments are required to determine whether specific transcription factors are involved in modulating rRNA gene expression in these systems.

Regulation of pre-rRNA processing The primary 45s rRNA transcript is processed in sequential steps into 18S, 28S, and 5.8s rRNAs prior to their incorporation into large and small ribosomal subunits (reviewed


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by Hadjiolov 1980). snRNPs are involved in splicing and processing reactions required for mRNA maturation. Recent findings suggest that similar complexes may also be necessary for processing of the 45s rRNA precursor. In yeast, snRNAs, localized in the nucleolus, are associated with various rRNA precursors and implicated in rRNA maturation (Riedel et al. 1986; Tollewey 1987). Cells lacking snlO exhibit impaired growth and are defective in processing the 35s rRNA intermediate (Tollervey 1987). Similarly, cells in which the gene coding for U3 RNA, also designated snR17, has been deleted are not viable (Hughes et al. 1987). Yeast U14 snRNA, previously identified as snR128, is also involved in rRNA maturation, specifically influencing the accumulation of 18s rRNA relative to 25s rRNA (Li et al. 1990). Loss of U14 snRNA results in a significant reduction in 18s rRNA production, possibly owing to impaired processing of precursors containing 18s rRNA coupled with rapid turnover of unstable intermediates. Structurally related snRNAs, found in higher eucaryotes, are thought to possess similar functions in ribosome biogenesis. In mammalian cells, snRNAs U3, U8, and U13 have been found associated with preribosomal RNP complexes and are presumed to function by base pairing with specific sequences in the 45s rRNA transcript (reviewed in Maser and Calvet 1989; Tyc and Steitz 1989). It has been demonstrated that the 5' end of U3 RNA can be cross-linked in vivo to the rat pre-rRNA ETS (Stroke and Weiner 1989) and that processing of this region requires the U3 snRNP (Kass et al. 1990). Other potential sites of interaction between U3 RNA and the 45s pre-rRNA have also been reported. Development of in vitro assays to monitor prerRNA processing has permitted more detailed analysis of the involvement of snRNPs and additional protein factors. This assay has led to the identification of a heat-labile, micrococcal nuclease-insensitive protein factor essential for cell-free pre-rRNA processing (Mishima et al. 1988). In addition, the transcription termination factor TTFI also appears to have a role in processing of this transcript (Kuhn and Grummt 1989), although the termination and processing events can be uncoupled (Labhart and Reeder 1990). The function of snRNA in pre-rRNA processing is apparently associated with the rRNA precursor molecule itself in some lower eucaryotes. In Neurospora and Tetrahymena, the rRNA precursors can be self-spliced in vitro, a reaction dependent on specific sequences contained within the primary transcript (Inoue et al. 1986). The evolutionary significance of this self-splicing mechanism and its relation to higher eucaryotes is unknown. The possible involvement of U3 RNA in the regulation of pre-rRNA processing is supported by variations in the level and relative distribution of U3 RNA that occur in response to changes in the rate of rRNA synthesis. The level of U3 RNA is significantly reduced relative to other major snRNAs in nucleated frog and chicken erythrocytes which do not synthesize rRNA (Hellung-Larsen and Frederiksen 1977; Frederiksen and Hellung-Larsen 1979). Whether there is a direct link between the level of U3 RNA and the ability to process pre-rRNA has not yet been established. The level of rRNA can be regulated by degradation of pre-rRNA, thereby reducing the formation of 18s and 28s rRNA. This mechanism has been observed to operate in resting human lymphocytes, but is eliminated following phytohemaglutinin stimulation (Cooper 1973). Similar

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results have been observed in resting as compared with serum-stimulated mouse fibroblasts (Johnson et al. 1976), in senescent human fibroblasts as compared with proliferating cells (Wolf et al. 1980), in embryonic quail myofibres (Bowman and Emerson 1977), and after heat shock of various organisms, including cultured Drosophila cells (Bell et al. 1988a) and Xenopus oocytes (Labhart and Reeder 1987). Expression of 5S RNA genes The genes for 5s rRNA, unlike those for the 45s rRNA precursor, are located outside the nucleolus. The newly synthesized 5s rRNA must, therefore, be transported to the nucleolus where, in combination with r-proteins, it is assembled into the large ribosomal subunit prior to its exit from the nucleus. Transcription of the 5s rRNA gene by RNA polymerase I11 (reviewed by Geiduschek and TocchiniValentini 1988) is under the control of intragenic sequences, consisting of two functional control regions that bind three transcription factors designated TFIIIA, TFIIIB, and TFIIIC. Only TFIIIA is specific for 5s rRNA gene transcription, binding to both the intermediate ( + 67 to + 72) and box C ( + 80 to + 97) elements. TFIIIB and TFIIIC, which bind to box A ( + 50 to + 64), are shared by other genes transcribed by RNA polymerase 111. Other control regions, not required in vertebrate cells, have been identified in yeast, Neurospora, silkworm, and Drosophila 5 s rRNA genes (Geiduschek and Tocchini-Valentini 1988; Sharp and Garcia 1988; Challice and Segall 1989). Termination of 5 s RNA transcription requires a simple consensus sequence, consisting of a cluster of T residues surrounded by a GC-rich region. Termination occurs a few nucleotides downstream of the 3 ' terminus and the mature transcript is produced by a 3 ' exonucleolytic reaction (Xing and Worcel 1989a). While initial studies suggested transcription termination was independent of ancillary factors (Carey et al. 1986), a recent report indicates at least one termination factor is required (Gottlieb and Steitz 1989). How is 5 s rRNA transported from its site of synthesis to the nucleolus for assembly into the large subunit? In mammalian cells, a significant proportion of unbound 5s rRNA is found complexed with r-protein L5 (Steitz et al. 1988). Based on kinetic data and the cellular location of r-protein L5, Steitz et al. (1988) have proposed that newly synthesized 5s rRNA becomes associated with r-protein L5, which then functions in transporting the complex from the nucleoplasm to the nucleolus. One of the major systems used for studying regulation of 5s rRNA synthesis is the developing Xenopus oocyte, in which ribosomes are synthesized and stored for later use by the developing embryo. 5s rRNA is produced in large amounts at an early stage of oogenesis, well before ribosome assembly takes place, and stored as a 7 s RNP complex with TFIIIA (Picard and Wegnez 1979). The ability of oocytes to produce large quantities of 5s rRNA results from the transcription of the 5s rRNA multigene family, containing more than 20 000 individual genes, which are active only in developing oocytes (Wormington and Brown 1983; Wakefield and Gurdon 1983). Following fertilization, the oocyte-specific 5 s rRNA genes remain inactive. At the midblastula transition, 400 somatic 5s rRNA genes become active (Wormington and Brown 1983; Wakefield and Gurdon 1983). These genes differ from the oocyte-specific


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genes at six points in their nucleotide sequence (Peck et al. 1987), resulting in enhanced affinity for TFIIIA (Xing and Worcel 1989b). To explain the differential expression of the oocyte and somatic 5S rRNA genes, Wolffe and Brown (1988) suggest that disruption of the oocyte 5S rRNA gene transcription complexes by DNA replication results in their incorporation into chromatin, rendering them inaccessible to transcription factors (Wolffe and Brown 1988). In contrast, TFIIIA remains bound to the somatic genes and its presence prevents their association with histones. Alternatively, two forms of TFIIIA have been identified, both binding to the somatic 5s rRNA genes, but having different affinities for the oocyte 5s rRNA genes (Blanco et al. 1989). Modulation of TFIIIA gene transcription and the level of TFIIIA appears to play a primary role in regulating 5S rRNA synthesis (Scotto et al. 1989; Blanco et al. 1989). Viral infection of mammalian cells affects RNA polymerase I11 mediated gene expression, including the rate of 5S rRNA gene transcription. Following infection with adenovirus, the adenovirus Ela gene product increases the rate of 5s rRNA gene transcription by increasing the proportion of phosphorylated TFIIIC (Hoeffler et al. 1988). Since this factor is also required by other genes transcribed by RNA polymerase 111, the effect of the Ela protein is not restricted to 5S rRNA. The activities of TFIIIB and TFIIIC are, in contrast, inhibited by infection of cells with poliovirus (Fradkin et al. 1987). The changes in transcription of both 5S rRNA and the 45s rRNA precursor genes (Rubinstein and Dasgupta 1989), caused by poliovirus infection, may have a significant effect on ribosome biogenesis. Cessation of cell proliferation generally involves a depression in ribosome biogenesis and a corresponding decrease in 5S rRNA levels (Gokal et al. 1986; Tower and SollnerWebb 1988). During encystment in Acanthamoeba, the decrease in 5S rRNA gene transcription is accompanied by changes in the levels of gene-specific transcription factors (Imboden et al. 1989). Similarly, the transition of mouse L1210 cells from exponential to stationary phase results in a decrease in the rate of 5S rRNA gene transcription which is related to a specific reduction in TFIIIB (Tower and Sollner-Webb 1988). Levels of TFIIIA, TFIIIC, and RNA polymerase 111, in contrast, remain constant. During differentiation of F9 embryonal carcinoma cells, the decrease in RNA polymerase I11 directed transcription of genes, including 5s rRNA, tRNA, and middle repetitive elements, is also mediated by a reduction in the activity of TFIIIB (White et al. 1989). Since TFIIIB is required by many genes transcribed by RNA polymerase 111, these observations suggest that this transcription factor modulates synthesis of these genes under a variety of physiological conditions. TFIIIA has the ability to bind to either the 5S rRNA gene or its RNA product. Thus, synthesis of 5S rRNA in vitro is inhibited by the addition of 5s rRNA, presumably by a feedback inhibition (Gruissem and Seifart 1982). Brow and Geiduschek (1987) have demonstrated that, in Saccharomyces cerevisiae, the inhibition resulting from addition of 5S rRNA is due to competition between RNA and DNA for a transcription factor and is correlated with the formation of a complex between r-protein YL3 and 5S rRNA. These authors have proposed that regulation of 5S rRNA synthesis involves competition between r-protein YL3 and TFIIIA for 5s rRNA. In the presence of a pool of r-protein YL3, which can bind to newly synthesized 5S rRNA, the

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resulting YL3 RNP is available for assembly into preribosomal subunits and subsequent migration to the cytoplasm. When free r-protein YL3 is limiting, TFIIIA binds 5S rRNA in competition with 5S rDNA, resulting in a decrease in transcription. This mechanism may also operate in Xenopus oocytes during formation of the 7S RNP. The reduction in 5S rRNA transcription observed following administration of cycloheximide to murine thymic lymphosarcoma cells argues for the involvement of a short-lived transcription factor (Gokal et al. 1986). Earlier studies using murine L cells treated with sufficient actinomycin D to block only 45s rRNA synthesis, however, indicated that 5s rRNA levels are regulated posttranscriptionally (Perry and Kelley 1968). Posttranscriptional mechanisms have also been reported to regulate 5S rRNA accumulation during decreases in ribosome production under diverse conditions (reviewed in Monier 1974). More recently, studies from this laboratory have demonstrated that, following terminal differentiation of rat L6 myoblasts, there is a drop in 5S rRNA accumulation that is unrelated to changes in the rate of transcription, but results from decreased stability of the newly synthesized 5S rRNA (Zahradka et al. 1989). This decrease parallels the reduction in 45s rRNA precursor gene transcription (Krauter et al. 1979; Jacobs et al. 1985). Similar observations have been made following glucocorticoid inhibition of P1798 T-lymphoma cell proliferation (Mahajan et al. 1989). Different mechanisms apparently operate to regulate the level of 5S rRNA under changing physiological conditions in eucaryotic cells. Ribosomal proteins Gene structure of r-protein Most r-protein genes are individually dispersed throughout the eucaryotic genome and not organized into closely linked transcriptional units (reviewed by Mager 1988). Their coordinate synthesis, thus, cannot be accounted for by autogenous regulation, as found in Escherichia coli. The organization of r-protein genes into operons in E. coli permits a coupling of r-protein synthesis with ribosome assembly, in which certain r-proteins synthesized in excess interact with their respective mRNA to block further translation of the mRNA. In addition, each repressor r-protein inhibits not only its own synthesis but also the synthesis of some, and in some cases all, of the r-proteins whose genes occur in the same transcriptional unit as the repressor protein (reviewed by Nomura et al. 1984). Since each eucaryotic r-protein gene is a single functional unit containing all the essential regulatory sequences, their balanced production may, therefore, involve certain common control elements. Extensive analysis has identified a number of conserved DNA sequences in more than 20 yeast r-protein genes (reviewed by Mager 1988). Each r-protein gene contains a TATA box, a TC-rich region, and two UAS. Two common UAS elements, originally designated HOMOL-1 and RPG box, share several nucleotide positions. The yeast TATAbox binding factor TFIID, essential for transcription of most genes by RNA polymerase 11, is presumed to bind to the r-protein gene promoter (Horikoshi et al. 1989). The TCrich region, identified in most yeast r-protein genes, is also present in other yeast genes capable of high levels of expression (Struhl 1987). In many r-protein genes, these sequence motifs are required for maximum transcription (Rotenberg and Woolford 1986; Woudt et al. 1987). Although the UAS


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elements have also been identified in genes other than those coding for the r-proteins, they are largely r-protein gene specific and organized in tandem pairs, usually 250-450 nucleotides upstream of the transcription initiation start site (Planta and Raue 1988). The RPG-box consensus sequence AACACCCATACATTT is present in both elements and binds a single 150-kDa protein called TUF (Vignais et al. 1987). This protein coordinates transcription of all yeast r-protein genes except L3 and S33. While the sequences required for binding TUF are absent in L3 and S33 r-protein genes, another trans-acting factor, independently designated TAF or SUF, binds to the UAS element of these genes (Vignais et al. 1987; Hamil et al. 1988). Since their expression remains coupled with other r-protein genes, additional transacting factors must exist to coordinate the expression of r-protein genes in yeast. Hamil et al. (1988) identified an 82-kDa protein that interacts with UAS elements of r-protein genes binding either TUF or TAF. Since TAF interacts with other growth-regulated genes, this transcription factor may also be involved in coordinating gene expression with cell proliferation (Dorsman et al. 1989). The r-protein genes of Xenopus, Drosophila, and mouse share a common feature, a pyrimidine-rich region flanking the transcription initiation site, which is absent from yeast r-protein genes. While both TATA- and CAAT-like sequences are present in Drosophila r-protein genes (O'Connell and Rosbach 1984), they are found at unusual locations, an observation that implies the TATA boxbinding factor TFIID may not be required for their transcription. An analogous suggestion can be made for the murine and Xenopus r-protein genes that completely lack the TATA box (Mager 1988). The r-protein genes of Drosophila, Xenopus, and mouse also have little homology with other RNA polymerase I1 directed gene promoter elements. Preliminary studies have identified several proteinbinding regions in the r-protein L14 gene promoter of Xenopus that are essential for transcription (Carnevali et al. 1989). Similarly, the murine r-protein L32 gene promoter contains five protein-binding domains required for maximum gene expression within a 150- to 200-bp region that spans the transcription start site (Atchison et al. 1989). At least one cis-acting element, present in the r-protein L32 and L30 (but not S16) gene promoters, binds a common transcription factor (Hariharan et al. 1989). Whether these or other promoter elements are involved in coordinating r-protein mRNA synthesis has not been examined. Mammalian r-protein genes are present in multiple copies, but only one contributes to the synthesis of r-protein mRNA (Monk et al. 1981). The murine r-protein L32 gene family contains a single transcriptionally active gene and 15 pseudogenes (Dudov and Perry 1984). The characteristic that distinguishes pseudogenes from the transcriptionally active gene is the absence of introns, which may have arisen by integrating DNA synthesized from partially processed RNA transcripts by reverse transcriptase (Dudov and Perry 1984). Other mammalian r-protein gene families such as L18, L30, L35a, S16, and S18 display a similar gene organization (Wiedemann and Perry 1984; Peled-Yalif et al. 1984; Chen and Roufa 1988; Wagner and Perry 1985; Kuzumaki et al. 1987). Regulation of r-protein gene transcription Under steady-state growth conditions, equimolar amounts of each r-protein are produced, irrespective of gene copy

69, 1991

number. In yeast, most r-proteins genes occur in duplicate, although a few are present in only one copy (reviewed by Mager 1988). For genes present in duplicate, each copy contributes to the total amount of mRNA present in the cell, even though their levels of expression may differ significantly (Abovich et al. 1985; Leer et al. 1985). A mechanism that compensates for naturally occurring differences in gene copy number must, therefore, be part of the overall transcription control Drocess. prodiction of approximately 75 different yeast r-proteins is coordinately regulated under a variety of physiological conditions, primarily at the level of transcription. In response to changes in growth conditions after a nutritional upshift (Donovan and Pearson 1986; Herruer et al. 1987) or following heat shock (Kim and Warner 1983; Herruer et al. 1988), the rates of r-protein gene transcription vary coordinately. Details of the mechanisms and potential transacting factors associated with this mode of regulation, however, have not been reported. One of the few examples of transcriptional control of r-protein genes in mammalian cells has been reported during differentiation of cultured mouse MM14 myoblasts, in which a downshift in r-protein production results, in part, from a decline in the rate of transcription (Agrawal and Bowman 1987). The lower steady-state level of r-proteins also involves a decrease in the translational efficiency of r-protein mRNAs. Likewise, r-protein gene transcription is stimulated in the liver of rats treated with glucocorticoids, but a corresponding increase in r-protein mRNA levels fails to occur (Flusser et al. 1989). Transcriptional control of r-protein production does not appear to be a common regulatory mechanism in mammalian systems. Posttranscriptional control of r-protein synthesis Production of r-protein is regulated during development of Xenopus oocytes and embryos following synthesis of the primary transcript. In anucleolate mutants of Xenopus, normal levels of r-protein mRNA accumulate during early embryogenesis, in spite of the absence of rRNA synthesis (Pierandrei-Amaldi et al. 1982). At the stage of embryogenesis when r-protein mRNA is first translated and r-proteins accumulate, a specific decrease in r-protein mRNA occurs, resulting from transcript instability. These authors suggest that this temporal correlation between the onset of r-protein production and the decrease in mRNA stability stems from a type of autogenous control in which the excess r-proteins affect the stability of their own transcripts. Studies involving transformation or injection of extra copies of r-protein genes or mRNAs into eucaryotic cells have provided additional evidence for posttranscriptional regulation of r-protein production. In Xenopus oocytes, an increase in the gene dosage for r-protein L1 fails to induce unbalanced production of r-protein L1. Rather, the injected genes are transcribed, but their processing is incomplete, leading to accumulation of an unstable precursor RNA transcript that contains two of its nine introns (Caffarelli et al. 1987). To determine whether the excess free r-protein Ll inhibited processing of excess transcripts, antibodies against r-protein L1 were microinjected along with extra c o ~ i e sof the r- rotei in L1 gene (Pierandrei-Amaldi et al. 1988). sequestration of the-free protein by the antibodies augmented processing of the corresponding transcripts and production of mature r-protein L1 mRNA. Since free


REVIEW / SYNTH~SE

r-protein is absent in fertilized eggs, microinjection of the cloned r-protein L1 gene resulted in the accumulation of excess mRNA (Pierandrei-Amaldi et al. 1988). This mechanism also operates for a few r-proteins in yeast (Bromley et al. 1982; Warner et al. 1985). Introduction into yeast cells of a high copy number plasmid containing the r-protein L32 gene results in the accumulation of r-protein L32 precursor RNA, but not its mRNA, which suggests the protein may bind to its own primary transcript and prevent splicing (Dabeva et al. 1986). This posttranscriptional mechanism for regulating yeast r-protein levels in response to an increase in gene dosage is not common. Control of r-protein production through processing of primary RNA transcripts has not yet been documented in mammalian cells.

Translational control of r-protein synthesis Variations in distribution of r-protein mRNAs between polysome and free mRNPs following changes in physiological conditions suggest that translational regulation may play a major role in modulating r-protein production. It has been established that storage of r-protein mRNA in free mRNPs renders the mRNA unavailable for translation (reviewed in Larson and Sells 1987). Redistribution of r-protein mRNAs occurs under a variety of physiological conditions in higher eucaryotes. Treatment of lymphosarcoma cells with dexamethasone, a glucocorticoid analogue, causes a reduction in rRNA synthesis. while the rate of r-wotein mRNA formation remains unchanged (Meyuhas e; al. 1987). The proportion of polysome-associated r-protein mRNAs, however, drops relative to other mRNAs, causing an overall decrease in r-protein synthesis. The r-protein mRNA redistribution may reflect translational control, but whether the preexisting r-protein mRNAs actually shift between the free and polysomeassociated mRNA populations has yet to be established. Similarly, serum stimulation of mouse fibroblasts causes a change in the distribution of r-protein mRNA between free mRNPs and polysornes, increasing the proportion present in the polysome fraction (Geyer et al. 1982; Kaspar et al. 1990). Recently, a direct correlation was observed between phosphorylation of eIF-4E, the mRNA cap binding protein, and redistribution of r-protein mRNA (Kaspar et al. 1990). Activation of translation initiation factors by modifications such as phosphorylation may thus have an important role in recruiting r-protein mRNAs into polysomes. The r-protein mRNAs are also stored as inactive mRNPs during early Drosophila embryogenesis. Although the relative abundance remains constant, r-protein mRNAs are associated with polysomes during oogenesis and late embryogenesis, but are absent during early embryogenesis (Al-Atia et al. 1985). During these periods of development, r-protein synthesis parallels transcription of rRNA. In mutants with a reduced number of rRNA genes and thus apparently lower levels of rRNA synthesis, neither r-protein mRNA levels nor their translation are altered (Kay and Jacobs-Lorena 1985), suggesting that the rRNA concentration fails to influence translational regulation of r-protein production during Drosophila development. Synthesis and accumulation of r-protein mRNA during development in Xenopus do not correspond to its utilization (Pierandrei-Amaldi et al. 1982; Baum and Worthington 1985). As oocytes mature, r-protein mRNA is dissociated from polysomes, deadenylated and degraded (Hyman and Wormington 1988). Sequences in the 3' terminus of

11

r-protein mRNA appear to direct both deadenylation and polysomal dissociation during oocyte maturation (Hyman and Wormington 1988). In contrast, r-protein mRNAs are synthesized and stored as free mRNPs during early embryo development in Xenopus and presumably recruited into polysomes at later stages (Pierandrei-Amaldi et al. 1982). Other mechanisms for regulating r-protein production have been described. Changes in r-protein formation during early development in Dictyostelium are unrelated to alterations in mRNA levels, but rather are controlled primarily at the level of translational initiation (Steel and Jacobson 1987). Increased r-protein gene dosage in yeast results in enhanced mRNA levels, but the mRNAs are translated inefficiently (Pearson et al. 1982; Warner et al. 1985). In Tetrahymena, r-protein synthesis ceases during starvation as a result of translational regulation (Andreasen et al. 1984). Recent studies have shown that the 5 ' untranslated region of the r-protein mRNA is involved in translational regulation during Xenopus development. Fusion of this region to an unrelated mRNA and microinjection of this construct into fertilized eggs results in translational regulation typical of r-proteins during embryo development (Mariottini and Amaldi 1990). Whether structural regions of r-protein mRNAs in other eucaryotes contain similar regulatory regions has not as yet been determined.

Synthesis of r-protein and posttranslational control The abundance of r-proteins in eucaryotic cells may not reflect the amount of translationally active mRNAs. In yeast, an increase in the r-protein L29 gene number causes a proportional increase in its mRNA without a corresponding change in protein concentration, since the newly synthesized r-protein L29 is rapidly degraded (Warner et al. 1985). Similar studies have confirmed that this mode of regulation operates for other yeast r-proteins (Elbaradi et al. 1986; Maicas et al. 1988; Tsay et al. 1988). The normal level of r-proteins in this system is maintained, therefore, by rapid degradation of the r-protein following translational spillover. In the absence of rRNA synthesis or following a reduction in the rate of rRNA transcription, a similar mechanism modulates r-protein levels in higher eucaryotes. The r-proteins are synthesized at normal rates, but subsequently degraded in anucleolate mutants of Xenopus (PierandreiAmaldi et al. 1985), following terminal differentiation of rat myoblasts (Krauter et al. 1980; Jacobs et al. 1985), in regenerating rat liver (Tsurugi and Ogata 1979), and following inhibition of rRNA synthesis in cultured animal cells by actinomycin D (Craig and Perry 1971; Warner 1977; DePhilip et al. 1980). Increasing the gene dosage of r-proteins S16 and L32 in mouse myoblasts results in the accumulation of the corresponding mRNAs to levels that are directly proportional to the gene copy number (Bowman 1987b). The overproduced r-proteins are then rapidly degraded. Transcription of human r-protein S14 DNA, transfected into hamster cells, also causes elevated mRNA levels but r-protein S14 accumulation does not increase (Rhoads and Roufa 1987). Similarly, injection of polyadenylated r-protein L1 mRNA into Xenopus oocytes induces an increase in r-protein L1, which is rapidly degraded (Baum et al. 1988). Although regulation of r-protein production in response to an artificial situation, such as an


12

BIOCHEM. CELL BIOL. VOL. 69, 1991

FIG. 1. Ultrastructure of the nucleolus consisting of the granular component (g), fibrillar centre Cfc), dense fibrillar component (arrowheads), and associated chromatin (c). Bar = 0.5 am. increase in gene dosage, occurs by translational spillover, this mechanism also operates in higher eucaryotes under a variety of physiological conditions. Why under one set of physiological conditions translational spillover takes place, while under another mRNA redistribution occurs is unclear. The nucleolus The role of the nucleolus in ribosome biogenesis The nucleolus was established as the site of rRNA synthesis by the classical experiments in the laboratories of Birnsteil, Perry, and Gurdon using either anucleolate mutants of Xenopus and Drosophila or microbeam irradiation of this structure (reviewed by Sollner-Webb and Tower 1986). Nucleoli, representing a complex organization of chromatin consisting of rRNA gene clusters, form at special chromosomal sites termed NORs. The nucleolar ultrastructure, shown in Fig. 1, is characterized by ( i ) a granular zone, occupying the peripheral region of the nucleolus and containing ribosomal subunits at various stages of assembly; and ( i i ) a fibrillar zone, representing areas of active gene transcription, which is located in the central region of the structure and consists of one or more fibrillar centres surrounded by a dense fibrillar component. The newly transcribed rRNA molecules are immediately associated with specific ribosomal proteins within the granular component of the nucleolus to form the mature ribosomal subunits. RNA polymerase I has been immunocytochemically localized in the fibrillar centres, suggesting that these areas are sites of active RNA synthesis (Scheer and Rose 1984). During mitosis, RNA polymerase I remains associated with the NORs and, since RNA synthesis does not occur during this period, the enzyme must be maintained in an inactive state. Other factors such as CAMP-independent protein kinase NII, which phosphorylates RNA polymerase I and increases polymerase activity (Duceman et al. 1981), may

be implicated in modulating transcription. Additional components such as topoisomerase I and nucleolin (which may also be involved in rDNA transcription) are also phosphorylated by NII kinase and remain associated with rDNA in mitotic cells (Durban et al. 1985; Caizergues-Ferrer et al. 1987). The presence of these proteins presumably indicates which genes will be actively transcribed in the next interphase (Lapeyre et al. 1987). Although RNA polymerase I and rDNA are localized in the fibrillar centres of the nucleolus, autoradiographic studies of rDNA transcription have shown that label is predominantly associated with the surrounding dense fibrillar component (h4irre and Stahl 1981). To resolve these contradictory observations, Wachtler et al. (1989) reinvestigated the function of the fibrillar centre and dense fibrillar component using human Sertoli cells in which the nucleolar components are distinct and spatially arranged in a specific pattern. Using in situ hybridization, silver staining, and autoradiography, these authors concluded that the site of rDNA transcription in these cells is the dense fibrillar component. Although the role of the fibrillar centre has yet to be elucidated, its close association with the dense fibrillar component, as well as its high concentration of RNA polymerase I and other specific nucleolar proteins, suggests that this structure contains proteins necessary for rRNA synthesis or subunit assembly (Wachtler et al. 1987). The nucleolus also contains a fine network of 4-nm filaments, whose major component is a 140-kDa protein, which forms a scaffolding distinct in both its organization and composition from the nuclear matrix (Franke et al. 1981). Although the function of nucleolar scaffolding is unknown, it may provide structural support for the spatial arrangement of transcriptionally active rRNA genes or assembly of ribosomal subunits (Franke et al. 1981). It is interesting to note that these structures are absent in cells

REVIEW /


such as nucleated erythrocytes, mitotic cells, and spermatocytes which are inactive in rRNA synthesis, while present in somatic cells and Xenopus oocytes which have high levels of rRNA transcription (Franke et al. 1981; Benavente et al. 1984).

The role of chromatin structure in nucleolar formation and function rRNA synthesis, while essential for nucleolar formation, is not necessarily a primary determinant. Although formation of nucleoli can be inhibited when sequences extrinsic to the NOR are disrupted by chromosome rearrangements or laser microirradiation, other cytogenetic experiments indicate that it may be an intrinsic property of the rDNA to organize a nucleolus (summarized in Karpen et al. 1988). Is tandem repetition of active rDNA genes required for nucleolus formation? Karpen et al. (1988) demonstrated that insertion of a transcriptionally active rRNA gene into Drosophila euchromatin (non-rDNA chromosomal regions) resulted in the formation of mininucleoli, suggesting that neither tandem gene repetition nor heterochromatic location is an absolute requirement for rRNA synthesis. Molecular studies, however, suggest that tandem repetition of rDNA genes may play a role in optimizing rRNA transcription by permitting RNA polymerase I to continue from one gene to the next (Baker and Platt 1986), enabling a rapid response to physiological changes. The variation in number and structural organization of nucleoli is reflected by different levels of transcriptional activity (reviewed in Goessens 1984). An increase in the number of nucleoli may result from activation of previously inactive NORs. Using the technique of in situ hybridization, Wachtler et al. (1986) observed several patches of rDNA genes scattered throughout the nucleus, clearly in excess of the number of nucleoli present in resting lymphocytes. Following stimulation by phytohemaglutinin, the number of separate, distinct patches decreased significantly while the number of nucleoli increased slightly, suggesting that previously inactive NORs, in becoming transcriptionally active, tend to associate with one another. During this process, a reorganization must take place in which movement and (or) conformational changes of the chromatin structure occurs. Recent observations with Friend cells suggest, however, that the rDNA chromatin structure is maintained throughout the cell cycle, even though the rate of rDNA transcription drops significantly during mitosis (Conconi et al. 1989). Nucleolin, a multifunctional nucleolar protein Almost two decades ago, Busch and his co-workers identified C23, a 110-kDa protein later named nucleolin, as a major nucleolar protein (Orrick et al. 1973). Considering its unusual structure and activities, several groups have suggested that nucleolin is a multifunctional protein involved in ribosome biogenesis. The N-terminal region of nucleolin contains four highly acidic regions interspersed with basic lysine residues (Lapeyre et al. 1987) and it has been postulated that this region interacts with the basic domains of histones and r-proteins. The homology of the N-terminal domain with the high mobility group proteins also suggests that nucleolin may bind to histone, causing decondensation of chromatin (Erard et al. 1988). Whether nucleolin also functions in recruiting rDNA int'o nucleoli or in regulating rRNA gene transcription is still unclear. The distinct structure of nucleolin, however, provides a basis for postulating

SYNTHBSE

13

how this molecule influences the equilibrium between active and inactive rRNA genes. The carboxy-terminus of the nucleolin molecule contains a glycine-rich domain in an extended conformation that may be involved in protein-protein interactions (Lapeyre et al. 1987). This sequence is also found in fibrillarin, a nucleolar protein thought to function in pre-rRNA processing through an association with specific U RNAs (Lischwe et al. 1985; Tyc and Steitz 1989; Schimrnang et al. 1989). Finally, a third area, located in a more central portion of the nucleolin molecule, binds RNA and contains four RNP consensus sequences present in various RNA-binding proteins (Bugler et al. 1987). The function of this region has not been determined, but it is interesting to note that nucleolin also binds preferentially to spacer sequences between repeated rRNA genes (Olson et al. 1983). Although distributed throughout the nucleolus, nucleolin is predominantly localized in the dense fibrillar component (Spector et al. 1984). In addition, most nucleolin is associated with newly synthesized pre-rRNAs and not with preribosomal complexes (Herrera and Olson 1986). Nucleolin levels are also correlated with the rate of rRNA synthesis (Orrick et al. 1973; Ballal et al. 1975; Bouche et al. 1987). For example, nucleolin concentrations are low in resting cells where the rate of rRNA synthesis is depressed, while it represents up to 5% of nucleolar proteins in exponentially growing cells (cited in Lapeyre et al. 1987). During ribosome biogenesis, both phosphorylation and proteolytic cleavage of nucleolin have been implicated in controlling the rate of pre-rRNA transcription (Bouche et al. 1984; Suzuki et al. 1985). Furthermore, synthesis and posttranslational modification of nucleolin precedes transcription of rDNA and synthesis of r-proteins during oogenesis and embryogenesis in Xenopus laevis (Caizergues-Ferrer et al. 1989). Inactivation of the protease(s) associated with maturation of nucleolin inhibits synthesis of pre-rRNA (Bouche et al. 1984), implying that proteolytic processing of nucleolin has a role in regulating rDNA transcription. Interestingly, a drop in the level of unprocessed protein by injection of an antibody to nucleolin into nuclei of Chironomus tentans salivary gland cells causes a significant increase in rRNA gene activity (Egyhazi et al. 1988). The authors suggest that inhibition of rDNA transcription by unprocessed nucleolin is prevented by binding of the antibody and this effect may be analogous to the role of proteolytic cleavage. A role for nucleolin in reinitiation of rRNA synthesis during the Go to GI transition in ABAE cells has been implied (Bouche et al. 1987). Stimulation of quiescent ABAE cells in serum-free medium containing only bFGF results in a significant increase in the nucleolar content of nucleolin. bFGF may stimulate rRNA synthesis by activating nucleolar CAMP-independentprotein kinase NII, which can phosphorylate nucleolin (Bouche et al. 1987). The enhancement of RNA polymerase I activity in isolated nuclei by bFGF may also result from an increase in phosphorylation of the enzyme by NII kinase (Bouche et al. 1987). Further study with other systems is required to firmly establish a role for nucleolin in ribosome biogenesis.

Other major nucleolar proteins Fibrillarin, a 34-kDa nucleolar protein specifically immunoprecipitated by autoimmune sera obtained from

14

BIOCHEM. CELL BIOL. VOL. 69,

1991

TABLE1. Control points in eucaryotic ribosome biogenesis


rRNA

r-protein

-

Cell type

Perturbation

Response

Cardiac myocytes Mouse MM14 myoblasts Rat liver Rat liver

Growth stimulation Insulin treatment Regeneration Glucocorticoid treatment

Increased Increased Increased Increased

Plants Acanthnmoeba Yeast Rat L6 myoblasts

Light exposure Encystment Heat shock Differentiation

Increased transcription Decreased transcription Decreased transcription Decreased transcription

P1798 lymphosarcoma cells P1798 lymphosarcoma cells P 1798 lyrnphosarcoma cells Mouse MM14 myoblasts HL-60 leukemia cells Ehrlich ascites cells Mouse L210 cells HeLa cells Tomato cell culture HeLa cells Xenopus oocytes

Glucocorticoid treatment

Decreased transcription

McDermott et al. 1989 Hammond and Bowman 1988 Dabeva and Dudov 1982 Dabeva and Ikonomova 1982; Frey and Seifart 1982 Tobin and Silverthorne 1985 Bateman and Paule 1986 Vienot-Drebot et al. 1989 Krauter et al. 1979; Jacobs et al. 1985 Cavanaugh and Thompson 1985

Cyclosporin treatment


Mahajan and Thompson 1987

Cycloheximide treatment


Gokal et al. 1986

Differentiation Differentiation Serum starvation Growth arrest Poliovirus infection Heat shock Heat shock Heat shock

Bowman 19870 Schwartz and Nilson 1988 Buttergeit et 01. 1985 Tower and Sollner-Webb 1987 Rubinstein and Dasgupta 1989 Nover et 01. 1986 Parker and Bond 1989 Labhart and Reeder 1987

Mouse fibroblasts

Growth arrest

Human lymphocytes

Growth arrest

Human fibroblasts

Growth arrest

Drosophila

Heat shock

Quail myoblasts

Differentiation

Decreased transcription Decreased transcription Decreased transcription Decreased transcription Decreased transcription Decreased transcription Decreased transcription Inhibits transcription termination, incomplete processing Incomplete processing, pre-rRNA degraded Incomplete processing, pre-rRNA degraded Incomplete processing, pre-rRNA degraded Incomplete processing, pre-rRNA degraded Incomplete processing, pre-rRNA degraded

Yeast

Nutritional upshift

Increased transcription

Rat liver Yeast

Glucocorticoid treatment Heat shock

Increased transcription Decreased transcription

Mouse MM14 myoblasts Xenopus oocytes

Differentiation Increased gene dosage

Yeast

Increased gene dosage

Yeast

rna mutations

Xenopus oocytes


P1798 lymphosarcoma cells Mouse fibroblasts

Glucocorticoid treatment

Decreased transcription Incomplete processing, transcript degraded Incomplete processing, transcript degraded Incomplete processing, transcript degraded Incomplete processing, transcript degraded mRNA redistribution

Serum stimulation

mRNA redistribution

Drosophila Xenopus Xenopus Yeast

Early embryogenesis Early embryogenesis Oocyte maturation Increased gene dosage

Dictyostelium

Early development

Yeast


Stored mRNA Stored mRNA mRNA degradation Decrease in translational initiation Decrease in translational initiation Excess r-proteins degraded

transcription transcription transcription transcription

Ref.

Johnson et al. 1976 Cooper 1973 Wolf et al. 1980 Bell et 01. 1988a Bowman and Emerson 1977 Donovan and Pearson 1986; Herruer et al. 1987 Flusser et al. 1989 Kim and Warner 1983; Herruer et al. 1988 Agrawal and Bowman 1987 Caffarelli et al. 1987 Warner et al. 1985; Dabeva et al. 1986 Bromley et al. 1982 Caffarelli et al. 1987 Meyuhas et al. 1987 Geyer et al. 1982; Kasper et a!. 1990 Al-Atia et al. 1985 Pierandrei-Amaldi et al. 1982 Hyman and Wormington 1988 Pearson et al. 1982; Warner et al. 1985 Steel and Jacobson 1987 Warner et al. 1985; Elbaradi et al. 1986; Maicas et al. 1988; Tsay et al. 1988

REVIEW I

SYNTHBSE

15

TABLE1 (concluded)


Cell type

5 s rRNA

Perturbation

Response

Ref.

Xenopus anucleolate mutants Xenopus oocytes Rat L6 myoblasts

Embryogenesis

Excess r-proteins degraded

Pierandrei-Amaldi et at'. 1985

mRNA microinjection Differentiation

Excess r-proteins degraded Excess r-proteins degraded

Rat liver Cultured animal cells

Regeneration Actinomycin D treatment


Mouse MM14 myoblasts CHO cells

Increased gene dosage Increased gene dosage


Baum et al. 1988 Krauter el al. 1980; Jacobs et al. 1985 Tsurgi and Ogata 1979 Craig and Perry 1971; Warner 1977; DePhilip et al. 1980 Bowman 19876 Rhoads and Roufa 1987

Xenopus

Oocyte maturation embryogenesis Adenovirus infection Cycloheximide treatment

Increased transcription

Wolfe and Brown 1988

Increased transcription Decreased transcription

Hoeffler et al. 1988 Gokal et al. 1986

Encystment Growth arrest Differentiation Poliovirus infection Differentiation Actinomycin D treatment Glucocorticoid treatment

Decreased transcription Decreased transcription Decreased transcription Decreased transcription Excess 5 s rRNA degraded Excess 5 s rRNA degraded Excess 5 s rRNA degraded

Imboden et al. 1989 Tower and Sollner-Webb 1988 White et al. 1989 Fradkin et al. 1987 Zahradka et al. 1989 Perry and Kelley 1968 Mahajan et al. 1989

HeLa cells P1798 lymphosarcoma cells Acanthamoeba Mouse fibroblasts F9 embryonal carcinoma HeLa cells Rat L6 myoblasts Mouse L cells P1798 lymphosarcoma cells -

-

patients with scleroderma, is present in nucleoli of a variety of cell types including mouse fibroblasts, lymphocytes, rat liver, yeast, and HeLa cells (Ochs et al. 1985). This protein is localized in both the dense fibrillar component and fibrillar centres, and remains associated with NORs in metaphase and anaphase. Both its cellular location and association with U3, U8, and U13 RNAs suggest that fibrillarin functions in rRNA gene transcription and (or) processing (Ochs et al. 1985; Lischwe et al. 1985; Tyc and Steitz 1989). The observation that fibrillarin is barely detectable in transcriptionally repressed nucleoli of peripheral blood lymphocytes provides additional support for the involvement of fibrillarin in transcriptional events in the nucleolus (Ochs et al. 1985). In addition, the yeast protein NOPI, a homologue of the mammalian fibrillarin, is essential for cell growth (Schimmang et al. 1989). Cells containing a disrupted copy of the gene coding for the NOPl protein are not viable. Other proteins specifically localized in the granular component include the abundant phosphoprotein B23 (Spector et al. 1984), No38 (the B23 equivalent in Xenopus, SchmidtZachmann et al. 1987), ribocharin (an acidic protein associated with precursors of the large ribosomal subunit), and r-proteins (Hugle et al. 1985). Unlike proteins localized in the fibrillar zone, B23 is absent from NORs during mitosis (Spector et al. 1984). Since B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity, these properties may facilitate the attachment of r-proteins and other RNA-binding proteins to rRNA (Dumbar et al. 1989). Borer et al. (1989) have recently demonstrated that B23 and nucleolin shuttle constantly between the nucleus and cytoplasm, suggesting that these nucleolar proteins not only function within the nucleus, but are also involved in translocation of ribosomal components across the nuclear envelope. The movement and accumulation of the 70-kDa hsp to the nucleolus after heat shock treatment is correlated with visible changes in nucleolar morphology in eucaryotic cells

(Pelham 1984). Nucleolar function is protected in pre-heatshocked cells that contain high levels of hsps, suggesting that these proteins bind to immature ribosomal subunits and aid in the reassembly process (Pelham 1986). Whether hsps are active in ribosome biogenesis has not been established. Ribosome assembly and turnover Accumulation of ribosomes within a cell depends on the balance between the rate of subunit assembly and degradation. Ribosomal subunits are made in discrete stages within the granular component of the nucleolus. Initially, the 45s rRNA precursor associates with both 5s rRNA and r-proteins forming a complex referred to as an 80s preribosome (reviewed by Hadjiolov 1980). Maturation of the preribosome parallels the processing of the primary rRNA transcript, suggesting a close link between these two events. The ordered addition of r-proteins in the maturation process also appears to be essential for continued assembly (Kruiswijk et al. 1978; Todorov et al. 1983). For example, alterations in the amounts of either the yeast r-protein L3 or L16 result in a deficiency of 60s ribosomal subunits (Nam and Fried 1986; Rotenberg et al. 1988). While a continuous supply of r-proteins is necessary for ribosome maturation, only the final step in pre-rRNA processing is a critical point leading to mature ribosomal subunits (Dudov et al. 1978). Mature ribosomal subunits must travel from the nucleus into the cytoplasm. Selectivity in transport has been documented by the inability of mature procaryotic ribosomes to enter the cytoplasm following injection into the nucleus of Xenopus oocyte. The saturation kinetics associated with this process demonstrate the presence of a distinct transport system (Khanna-Gupta and Ware 1989). Association of specific r-proteins may serve as signals for transport, since subunits that are complete except for their final complement of r-proteins are retained in the nucleus (Lonn and Edstrom 1977).

BIOCHEM. CELL BIOL. VOL. 69, 1991 inactive rRNA genes

active rRNA genes

1

transcription inactive r-protein genes


18S, 28S, 5.8s rRNA

1

assembled subunits r-protein transcript

)C~--.

degradation

5 s rRNA

inactive 5 s rRNA genes

,

t

--%

degradation

degradation', \

transcription

--/

processing

r-protein mRNA

active 7 5 s rRNA genes

CYTOSOL

r-proteins translation turnover

I

I stored

r-protein

FIG. 2. Schematic representation of the regulatory steps in ribosome biogenesis. The cellular location for some of these processes is not known.

Mature ribosomes are stable complexes with half-lives ranging from 4.5 days in rat liver (Tsurugi et al. 1974) to more than 10 days in cultured L cells (Nissen-Meyer and Eikom 1976). Turnover thus plays a major role in maintaining appropriate levels of ribosomes in cells that have reduced rates of proliferation (Mader 1988). A drop in the demand for ribosomes observed in resting lymphocytes or following myogenesis results in decreased rates of rRNA processing and enhanced degradation of not only the rRNA precursor but also the entire preribosome (Cooper 1973; Clissold and Cole 1973; Bowman and Emerson 1977). Although the mechanisms involved in signalling an increase in turnover rate are not known, ubiquitin has recently been proposed to play a role in controlling ribosome stability. Yeast, Dictyostelium, and mammalian cells synthesize r-proteins fused to ubiquitin, which are incorporated into ribosomes (Finley et al. 1989; Redman and Rechsteiner 1989; MullerTaubenberger et al. 1989). Finley et al. (1989) have demonstrated that the fused proteins not only enhance processing of 20s pre-rRNA during the final stage of ribosome assembly, but also assist the efficiency of r-protein incorporation. While the role of ubiquitin remains undefined, this protein may protect the ribosome from degradation, its removal serving as a signal to commence degradation. A similar role in determining r-protein stability has also been proposed (Finley et al. 1989), suggesting ubiquitin may be involved in degradation following translational spillover.

Summary The eucaryotic ribosome consists of more than 80 different ribosomal proteins and four RNA species. In addition to having the central role in translation, the ribosome represents a unique model system for studying gene expression, since each ribosomal component is synthesized in a distinct cellular location and transported into the nucleolus for assembly into mature 60s and 40s subunits. In proliferating cells, mechanisms exist to coordinate the balanced production of individual ribosomal components. Numerous model systems have been used to examine the various strategies developed for regulating ribosome formation, and thus its precursors, in response to changes in growth rate, differentiation, and environmental conditions (summarized in Table 1 and Fig. 2). Although the nucleolus contains a collection of unique proteins and snRNA molecules that are essential at specific stages in ribosome formation, the function of these components is not yet completely understood. An examination of various model systems suggests that the primary mechanism regulating the rate of ribosome production involves changes in the rate of rRNA gene transcription. The resultant increase or decrease in rRNA synthesis is modulated by the level and (or) modification of transcription factor TFIC. These modulations have been noted following a variety of physiological changes including hormone treatment, heat shock, tissue regeneration, and following various stages of development. As more information


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becomes available, TFIC may yield additional components which will improve our understanding of the transcription process and how it is regulated. Although rRNA gene transcription is generally modulated following perturbation of growth rate, the same is not true for the 5 s rRNA gene. In a number of instances there is a drop in the transcription rate that results from decreased activity in one of the transcription factors. Alternatively, the decreased levels of 5 s rRNA occur with no change in transcription rate, but from selective degradation of the newly synthesized 5 s rRNA molecules. Our understanding is, at present, insufficient t o explain why one mechanism is preferred over the other. Diverse strategies have also been devised by the cell t o regulate the levels of r-proteins. In yeast, the r-protein levels are transcriptionally regulated in response t o physiological changes. In artificial situations in which the dosage of a particular r-protein gene is increased by transfection, the additional gene copies are transcribed and the resultant mRNAs are translated. The excess r-proteins are subsequently degraded. While this mechanism usually operates t o remove overproduced r-proteins, at least one example has been reported in which incomplete processing of pre-mRNA modulates the accumulation of the gene product. In higher eucaryotic systems, the production of r-proteins is generally regulated posttranscriptionally. For example, translational control operates through a redistribution of r-protein mRNAs between the polysomes and free mRNPs. Modulation of r-protein levels can also occur by rapid degradation of newly synthesized r-proteins produced in excess. The genes coding for each class of ribosomal component are transcribed by different RNA polymerases, primarily distinguished by their sensitivity t o a-amanitin and their specificity for certain gene promoters. While structurally distinct, three common subunits present in each polymerase species may function in coordinating transcription in yeast (Woychik et al. 1990). During balanced exponential growth, these common subunits may provide a mechanism for coordinating ribosomal precursor synthesis. Although our understanding of the processes regulating ribosome biogenesis is expanding, further investigations are needed to define the mechanisms that couple cell metabolic activities with the synthesis of ribosomal precursors (especially rRNA), and assembly of the ribosome and its degradation.

Acknowledgements Studies from this laboratory were supported by grants from the Medical Research Council (Canada) and the Natural Sciences and Engineering Research Council. The initial draft of this review was completed by B.H.S. during a visit t o the laboratory of Dr. Margaret Buckingham, Institut Pasteur, Paris, t o whom this author wishes t o express his thanks. The authors also thank Dr. Franz Wachtler for his electron micrograph of the nucleolus, and Nancy Sienna and Nadia Pece for critically reading this manuscript. ABOVICH, N., GRITZ,L., TUNG,L., and ROSBASH, M. 1985. Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 5: 3429-3435. AGRAWAL, M.G., and BOWMAN, L.H. 1987. Transcriptional and translational regulation of ribosomal protein formation during mouse myoblast differentiation. J. Biol. Chem. 262: 4868-4875.

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Ribosome biogenesis in replicating cells: Integration of experiment and theory.

Post-transcriptional regulation of ribosome biogenesis in yeast.

Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis.

Nom1 mediates pancreas development by regulating ribosome biogenesis in zebrafish.

Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

Endurance training lowers ribosome density despite increasing ribosome biogenesis markers in rodent skeletal muscle.

Eukaryote-specific rRNA expansion segments function in ribosome biogenesis.

Placeholder factors in ribosome biogenesis: please, pave my way.

RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis.

Discovery of a small molecule that inhibits bacterial ribosome biogenesis.

Bmi1 promotes erythroid development through regulating ribosome biogenesis.

Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis.

A liaison between mTOR signaling, ribosome biogenesis and cancer.

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle.

Ribosome Biogenesis is Necessary for Skeletal Muscle Hypertrophy.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

Tor1 and CK2 kinases control a switch between alternative ribosome biogenesis pathways in a growth-dependent manner.

Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis.

Ribosome-associated protein quality control.