1607235
Enzyme 1990;44:83-92
© 1990 S Karger AG, Basel 0013-9432/90/0444-0083S2.75/0
Translational Control of Ribosomal Protein Production in Mammalian Cells Robert P. Perry3, OdedMeyuhasb aInstitute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa., USA, and b Department of Developmental Biochemistry, Institute of Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem, Israel
Key Words. 5'-Un translated region • Ribosomal proteins ■ Polyribosomes • 5'-01igopyrimidine tract • Glucocorticoids Abstract. Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their under-representation in polyribosomes of normally growing cells. Recent studies have local ized the translational regulatory element to the 5' end of the rp mRNA and have demon strated that an oligopyrimidine tract, which adjoins the cap structure in all known vertebrate rp mRNAs, is an essential part of this element. Possible factors that might interact with the oligopyrimidine tract are discussed.
mechanism. Evidence for translational con trol of rp production has been observed dur ing developmental transitions of Dictyostelium discoideum [4], Drosophila melanogaster [5], Xenopus laevis [Amaldi and Pierandrei-Amaldi, this volume], and mouse myo blasts [6]. Selective regulation of rp mRNA translation also occurs when mammalian cells undergo a shift between growing and nongrowing states and during cellular re sponses to certain hormones and other stim uli [7-15]. In the present article, we will Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
The formation of eukaryotic ribosomes involves the coordinate production of four RNA components and more than 70 differ ent proteins. The synthesis of these ribo somal constituents is regulated in response to changes in physiological and develop mental states by a variety of mechanisms acting at all levels of gene expression from transcription to protein turnover [1-3]. In higher eukaryotes, the controlled translation of the mRNAs encoding ribosomal proteins (rp) is a particularly important regulatory
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General Features of rp mRNA Translation
The translational control of mammalian rp mRNAs was demonstrated by studies of their relative apportionment between trans lationally repressed mRNP particles and translationally active polyribosomes under various physiological and developmental conditions. In resting mouse fibroblasts, only about 20-30% of the rp mRNA is in a translationally active state, whereas in se rum-stimulated growing fibroblasts, this pro portion increases to about 65% [7, 9], Simi larly, in glucocorticoid-treated PI798 lym phosarcoma cells, which are arrested in growth and no longer produce ribosomes, the proportion of rp mRNA in polyribo somes is about 25 %, compared to about 65 % for exponentially growing cells [8, 10, 15]. In these latter studies, the apportionment be tween mRNP and polyribosomes was exam ined for 8 different rp mRNAs and various other mRNAs that encode general house keeping functions. Although the glucocorti coid treatment resulted in a marked decrease in the ribosome loading of 7 of the 8 rp mRNAs, the translation of the non-rp mRNAs was not significantly affected. Typi cal examples of such data are shown in fig ure 1. Clearly, there is some selectivity in the translational control of rp mRNAs under these conditions. Still another example of selective translational control of rp mRNA was observed in differentiating mouse myo blasts [6, 13]. In these cells, the proportion of
translationally active rp mRNA decreases from 45 to about 30% when myoblasts fuse into myotubes and increases to about 70% when myoblasts are treated with insu lin. In all of the studies cited above, it is clear that even when ribosomes are being synthe sized at maximum rates in rapidly proliferat ing cells, there is still a significant proportion of rp mRNA (about 30-40%) that is in a translationally repressed state. In contrast, only a minor fraction (< 10%) of most nonrp mRNAs is translationally repressed under the same conditions. Interestingly, a charac terization of cDNA clones for three mRNA species that are roughly equally distributed between free mRNP and polyribosomes in both mouse sarcoma 180 ascites cells and mouse erythroleukemia cells [16], revealed that one of them was an rp mRNA [17-19]. Although the other two mRNAs encode nonribosomal proteins, their extreme 5'ends bear a striking similarity to the rp mRNAs (see below). Another important feature of rp mRNA translation was deduced from a consider ation of the number of ribosomes associated with the translationally active fraction of rp mRNA in growing cells [8], As seen in the example of figure 1, the profiles of rp mRNAs in growing lymphosarcoma cells harvested 24 h after withdrawal of the hor mone are distinctly bimodal, suggesting two discrete populations of mRNA molecules which are distinguished by being either translationally inactive (free mRNP) or effi ciency translated (in polysomes containing 4-7 ribosomes). Considering the sizes of the rp mRNAs, this extent of ribosome loading indicates an average spacing of 31 codons per ribosome, which is similar to that of the non-rp mRNAs examined and believed to be Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
summarize the main features of translational control of rp mRNA in mammalian cells and try to provide some insight into the mecha nism^) responsible for this control.
Fig. 1. Translational control of rp mRNAs in PI798 lymphosarcoma cells. Cytoplasmic extracts of cells treated for 24 h with 10~7 M dexamethasone and either harvested immediately (Dexamethasone) or af ter incubation for an additional 24 h in the absence of hormone (Withdrawal) were fractionated on 15-45% sucrose gradients. RNA was extracted from each frac tion and aliquots representing equal volumes were dot blotted onto Nytran sheets. The blots were hy bridized with 32P-labelled probes specific for various rp mRNAs and autoradiographed. After melting off and decay of the rp signals, the blots were rehybrid ized with probes for selected nonribosomal mRNAs [actin, superoxide dismutase (SOD), glyceraldehyde-
85
3-phosphate dehydrogenase (GA3PD) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)] and autoradiographed again. Top two panels show the Ai60 profiles with the positions of the polyribosomes, monoribosomes (M) and 60S and 40S subunits indi cated. The other panels show the distributions of each mRNA species plotted as a percentage of the total of that species on the gradient. Open and solid circles denote rp mRNAs and non-rp mRNAs, respectively. The vertical dashed lines separate the polyribosome and subpolysome (free mRNP) fractions. Profiles from ‘withdrawal’ cells are indistinguishable from those of untreated cells [8].
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Table 1. Context of the AUG initiation codon in mammalian ribosomal protein mRNAs
C A G U Consensus rp General
-9
-8
-7
-6
-5
-4
-3
-2
-1
8 2
13 2 5 3
16 2
4 4 13 2
11
15 6 1
0 13 10 0
8 10 3 2
7 4 9 3
5 N G
C C
4 C C
G G
0 10 2 G C
C
1
c C
A G A G
A C
N
c
c
A
U
G
+4 3 6 13
A A
U____G U G
G G
The numbers in the table represent the frequency of occurrence of each nucleotide at the indicated position in a survey of 23 mammalian ribosomal protein mRNAs listed in Genbank. The tabulated sequences were as follows: h/mPo, hPl, rS8, rSlO, rS12, hS14, mS16, hS17, rS26, rL5, rL6, mL7, mL7a, rL18, rL19, rL21, rL26, mL30, rL31, mL32, rL34, rL36a and rL37. h, m and r signify human, mouse and rat proteins; S and L designate small and large subunit proteins, respectively.
changes in the content or activity of these ligands or of the general translation factors that compete with them.
Identification of the Translational Regulatory Element in rp mRNAs
The selective nature of the translational control of rp mRNAs suggests that they have some distinctive property that is recognized by the translational apparatus and/or by pro teins of the mRNP particles. A survey of the sequences spanning the AUG initiation co don in mammalian rp mRNAs (table 1 ) does not reveal any consistent pattern or trend that might be associated with an impairment of initiation efficiency. Indeed, the rp mRNAs examined to date conform to the general consensus and all contain a purine at the — 3 position, a feature that is believed to be especially important for efficient transla tion [21]. Although inverted repeat se quences capable of intrastrand base pairing Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
close to the theoretical maximum for any mRNA. Such bimodal distributions, which have been observed in other types of mam malian cells [6, 16] and in Xenopus embryos [Amaldi and Pierandrei-Amaldi, this vol ume], indicate that the rp mRNAs alternate between a repressed and an active state, and that, when in the active state, they are trans lated at near maximum efficiency. To account for the observed translational behavior of rp mRNAs, one may consider mechanistic models based on selective pack aging of rp mRNA into RNP particles [20] or the association of rp mRNA with a specific factor that limits its access to ribosomes and other components required for translation. Such ligands would presumably bind to the rp mRNAs with relatively high affinity and preempt interactions with translational fac tors. In the absence of ligands, the rp mRNAs are evidently capable of high effi ciency translation. The relative proportions of rp mRNA in translationally active and repressed states could be regulated by
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Table 2. 5' oligopyrimidine tracts in selected mammalian rp mRNAs rp mRNA
5' terminal sequence
mS16
CCUUUUCCG
mL32
CUUCUUCCUCG
mL30
CCUUUCUCG
mL7
CUCUCUUCUUUUCCG
Number of consecutive pyrimidines
References
8
23
10
24
8
25
14
26
*
mL7a
CUUUCUUUCUCCA
8/12
rL35a
CUCUUUCU
8
28
hS14
CUCUUUCCG
8
29
hS17
CCUCUUUUA
8
30
27
+
h, m and r refer to human, mouse and rat mRNAs, respectively. Two start sites detected. The center of a diffuse starting region.
are found in the 5'untranslated regions (UTRs) of some rp mRNAs, such repeats are not prevalent among all rp mRNAs. More over, the 5'UTRs of rp mRNAs are generally relatively short (25-50 nucleotides) and only one (L5) has been found to contain an AUG upstream of the true initiator codon [22], One common feature noted in all se quenced vertebrate rp mRNAs is an oligopy rimidine tract at the 5' terminus. In cases for which complete sequence information is available, this element is found to consist of a C residue at the cap site followed by an uninterrupted sequence of 7-13 pyrimidines (table 2). The fact that some non-rp mRNAs that exhibit the same translational control properties as rp mRNA also have a 5' termi nal oligopyrimidine tract [18, 19] suggests that this sequence may be a critical part of the translational regulatory element (TLRE).
Experiments designed to verify this con jecture and to localize the TLRE within the rp mRNAs have recently been carried out with PI798 lymphosarcoma cells, which, as described above, exhibit dramatic control of rp mRNA translation when exposed to the glucocorticoid hormone, dexamethasone [31]. In these experiments, the cells were transfected with chimeric genes capable of producing mRNAs in which rp and non-rp coding regions were linked in various combi nations to the 5'UTR of an authentic or mutated rp mRNA or to the 5'UTR of ßactin mRNA, a typical non-rp mRNA that is not translationally controlled (fig. 2). The apportionment of the chimeric mRNAs in polyribosomes and in free mRNP of hor mone-treated and control cells was deter mined and compared to that of an endoge nous actin and rp mRNA. The typical de crease in ribosomal loading of rp L32 mRNA Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
* +
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El ID •
pAct-L32 +74
-370 pAct-GH (74 n)
+167
266 pL30-GH (61 n)
l
D -179
+29
pS16-GH (29 n)
a +1
S16 wild-type
CCTTTTCC
pS16CM5-GH (27 n)
GAGTGACC I
pS16CM3-GH (27 n)
ACTTTTCC
Act-l
Act-L32 100-1
Ì-GH
S16CM5-GH 100
100
S16CM3-GH 100
80-
80 -
80
80-
60-
60 -
60-
60-
40-
40
40-
40
20-
20-
20-
CO
CD
E CO
>>
R
c
1
0 Con Dex
0
Con
Dex
0 Con Dex
o
b Con Dex
gradients and then analyzed as described in figure 1 for mRNAs produced by the transfected chimeric genes (hatched bars), the endogenous ß-actin gene (solid bars) and the endogenous rpL5 gene (open bars). The height of each bar represents the percentage of each mRNA that is in the polyribosome fraction.
in dexamethasone-treated cells was abol ished when its 5' end was replaced with that of ß-actin. Moreover, growth hormone (GH) mRNA acquired the translational control properties of an rp mRNA when its 5' end was replaced by that of rpL30 or rpS16, but
not when it was replaced by that of ß-actin. These results indicate that the 5' end of the rp mRNAs is necessary and sufficient to account for their translational control prop erties. Similar experiments, carried out with mouse myoblasts [Lewis Bowman, personal Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
Fig. 2. Localization of the translational control element in rp mRNA. a Schematic representation of the chimeric genes that were transfected into PI798 cells, b Cytoplasmic extracts of transfected cells, either untreated (Con) or exposed for 24 h to 10~7 M dexamethasone (Dex), were fractionated on sucrose
communication] or with X. laevis embryos [Amaldi and Pierandrei-Amaldi, this vol ume], have led to essentially the same con clusion. It is interesting to note that as few as 29 5'-terminal nucleotides of rpS16 are suffi cient to confer translational control on GH mRNA, thus localizing the TLRE to a rela tively short segment with no significant base pairing potential. Translational control was abolished when the chimeric genes were made with rpS16 mutants that cause the 5'-oligopyrimidine tract to be interrupted with purines or to be shortened by a couple of nucleotides. The loss of translational repression was demon strated by monitoring the polyribosomal as sociation of the mutant chimeric mRNAs (fig. 2), as well as by a direct immunoassay of the rate of synthesis of hGH [31]. These results indicate that the 5'-oligopyrimidine tract is a critical part of the TLRE and that either the terminal C residue, a minimal length of 7 or 8 consecutive pyrimidines or both of these features are essential for its activity. All of the rp 5'-terminal sequences known to date are consistent with this con clusion (table 2). There may also be impor tant structural information in the sequences immediately downstream of the pyrimidine tract. Although there are no obvious com mon sequence motifs in this region, there could conceivably be subtle patterns of nu cleotides that contribute importantly to the overall structure of the extreme 5' end.
Possible Trans-Acting Factors that Interact with the TLRE
Beyond a precise definition of the cis-act ing TLRE, it is also important to identify the trans-acting factors that determine the activ
89
ity of this regulatory element. Conceivably, there might be a factor that specifically binds to the TLRE and prevents the mRNA from interacting with ribosomes and other com ponents that are essential for protein synthe sis. An example of such a factor is the pro tein that represses the translation of ferritin mRNA by binding to a stem-loop structure in the 5TJTR of this mRNA [32], If an anal ogous TLRE-specific factor exists, it would presumably be quantitatively regulated or qualitatively modified in response to changes in cellular growth rate. An interac tion of protein(s) with the TLRE of mRNA that is sequestered in translationally inactive mRNP particles was suggested by a study of mouse P21 mRNA, a non-rp mRNA which has a 5'-terminal oligopyrimidine tract and which is subject to growth-dependent trans lational control [33]. Treatment of P21 mRNA with RNase T1 revealed that the G residues immediately downstream of the 5' pyrimidines are much more accessible when the mRNA is engaged in polyribosomes than when packaged in mRNP particles. Addi tional evidence for the possible involvement of such mRNP proteins in translational con trol has been discussed elsewhere [20]. In addition to a TLRE-specific factor, translational control may involve a factor that is part of the general protein-synthesiz ing machinery. Because the rp mRNAs nor mally exist in a delicate balance between translationally active and inactive states, they should be especially sensitive to changes in the content or activity of a critical translational initiation factor. If such a fac tor had a particularly low affinity for rp mRNA, decrease in its activity or content could lead to a selective diminution of rp mRNA translation. The fact that dexamethasone reduces the abundance of the mRNAs Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
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scription and turnover of the various rp mRNAs [7, 39], helps insure a well-balanced production of ribosomal proteins under a variety of physiological and developmental conditions.
References 1 Meyuhas O: Ribosomal protein gene expression in proliferating and nonproliferating cells; in Stein GS, Stein JL (eds): Recombinant DNA and Cell Proliferation. Academic Press, Orlando, 1984, pp 243-271. 2 Jacobs-Lorena M, Fried HM: Translational regu lation of ribosomal protein gene expression in eukaryotes; in Ilan J (ed): Translational Regula tion of Gene Expression, Plenum Press, New York, 1987, pp 63-85. 3 Mager WH: Control of ribosomal protein gene expression. Biochim Biophys Acta 1988;949:1 — 15. 4 Steel LF, Jacobson A: Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development. Mol Cell Biol 1987;7:965-972. 5 Al-Atia GR, Fruscolom P, Jacobs-Lorena M: Translational regulation of mRNAs for ribosomal proteins during early Drosophila development. Biochemistry 1985;24:5798-5803. 6 Agrawal MG, Bowman LH: Transcriptional and translational regulation of ribosomal protein for mation during mouse myoblast differentiation. J Biol Chem 1987;262:4868-4875. 7 Geyer PK, Meyuhas O, Perry RP, et al: Regula tion of ribosomal protein mRNA content and translation in growth-stimulated mouse fibro blasts. Mol Cell Biol 1982;2:685-693. 8 Meyuhas O, Thompson EA, Perry RP: Glucocor ticoids selectively inhibit translation of ribosomal protein mRNAs in PI798 lymphosarcoma cells. Mol Cell Biol 1987;7:2691-2699. 9 Kaspar RL, Rychlik W, White MW, et al: Simul taneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of euka ryotic initiation factor 4E after mitogenic stimula tion of Swiss 3T3 cells. J Biol Chem 1990;265: 3619-3622. Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
encoding the translational initiation factors eIF-4A, eIF-2a and eIF-4D by 60-70% in PI798 cells [15] is consistent with this idea. Moreover, an increase in the translational efficiency of rp mRNAs during transition of Swiss 3T3 cells from nongrowing to growing states occurs simultaneously with enhanced phosphorylation of the initiation factor elF4E [9], This factor, which is also known as the cap-binding protein, constitutes, to gether with eIF-4A and a 220-kD protein, the eIF-4F complex. Due to the relatively low abundance of eIF-4E [34], the entire elF4F complex is a limiting component in the binding of eukaryotic mRNAs to the ribo some. Since this step is generally considered to be the overall limiting step in translation [35], eIF-4F is a prime candidate for modu lating translation efficiency. Indeed, the lat ter has been implicated in discrimination between weak and strong mRNAs [for re view see Sonenberg, 36, and Rhoads, 37], Since rp mRNAs apparently have normal cap structures, as inferred from the heteroge nous products observed in SI nuclease pro tection analyses [23, 38], they should be able to bind eIF-4F. Thus, the novel 5'-terminal structure of the TLRE could define a binding site for specific inhibitory ligands and also help determine the ability of these ligands to be displaced by translational initiation factors. Irrespective of the exact mechanism, it would appear that the oligopyrimidine tract endows rp mRNAs and other mRNAs bear ing a similar 5' terminus with the property of being underutilized for translation, even un der optimal growth conditions, and of being exquisitely sensitive to conditions that ac company changes in growth rate. This trans lational control property, together with the remarkable equivalence in the rates of tran-
Translational Control of Ribosomal Protein Production
21 22
23
24
25
26
27
28
29
30
31
mRNPs and containing specific ScRNA and a characteristic set of proteins. EMBO J 1984;3:29— 34. Kozak M: The scanning model for translation: An update. J Cell Biol 1989;108:229-241. Tamura S, Kuwano Y, Nakayama T, et al: Molec ular cloning and nucleotide sequence of cDNA specific for rat ribosomal protein L5. Eur J Biochem 1987;168:83-87. Wagner M, Perry RP: Characterization of the multigene family encoding the mouse SI6 ribo somal protein: strategy for distinguishing an ex pressed gene from its processed pseudogene coun terparts by an analysis of total genomic DNA. Mol Cell Biol 1985;5:3560-3576. Dudov KP, Perry RP: The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell 1984;37:457— 468. Wiedemann LM, Perry RP: Characterization of the expressed gene and several processed pseudo genes for the mouse ribosomal protein L30 gene family. Mol Cell Biol 1984;4:2518-2528. Meyuhas O, Klein A: The mouse ribosomal pro tein L7 gene: Its primary structure and functional analysis of the promoter region. J Biol Chem 1990;265:11465-11473. Huxley C, Williams T, Fried M: The mouse rpL7a gene is typical of other ribosomal protein genes in its 5' region but differs in being located in a tight cluster of CpG-rich islands. Nucleic Acids Res 1990;18:5353-5357. Kuzumaki T, Tanaka T, Ishikawa K, et al: Rat ribosomal protein L35a multigene family: molec ular structure and characterization of three L35arelated pseudogenes. Biochim Biophys Acta 1987; 909:99-106. Rhoads DD, Dixit A, Roufa DJ: Primary struc ture of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 1986;6:2774— 2783. Chen I-T, Roufa DJ: The transcriptionally active human ribosomal protein S17 gene. Gene 1988; 70:107-116. Levy S, Avni D, Hariharan N, et al: The oligopyrimidine tract at the 5' end of mammalian ribo somal protein mRNAs is required for their trans lational control. Proc Natl Acad Sci USA 1991,88: 3319-3323. Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
10 Meyuhas O, Baldin V, Bouche G, et al: Glucocor ticoids repress ribosome biosynthesis in lympho sarcoma cells by affecting gene expression at the level of transcription, posttranscription and trans lation. Biochim Biophys Acta 1990;1049:38-44. 11 DePhilip RM, Rudert WA, Lieberman I: Prefer ential stimulation of ribosomal protein synthesis by insulin and in the absence of ribosomal and messenger ribonucleic acid formation. Biochemis try 1980;19:1662-1669. 12 Ignotz GG, Hokari S, DePhilip RM, et al: Lodish model and regulation of ribosomal protein synthe sis by insulin-deficient chick embryo fibroblasts. Biochemistry 1981;20:2550-2558. 13 Hammond ML, Bowman LH: Insulin stimulates the translation of ribosomal proteins and the tran scription of rDNA in mouse myoblasts. J Biol Chem 1988;263:17785-17791. 14 Schmidt T, Chen PS, Pellegrini M: The induction of ribosome biosynthesis in a nonmitotic secre tory tissue. J Biol Chem 1985;260:7645-7650. 15 Huang S, Hershey JWB: Translational initiation factor expression and ribosomal protein gene ex pression are repressed coordinately but by differ ent mechanisms in murine lymphosarcoma cells treated with glucocorticoids. Mol Cell Biol 1989; 9:3679-3684. 16 Yenofsky R, Cereghini S, Krowczynska A, et al: Regulation of mRNA utilization in mouse erythroleukemia cells induced to differentiate by expo sure to dimethyl sulfoxide. Mol Cell Biol 1983;3: 1197-1203. 17 Krowczynska AM, Coutts M, Makrides S, et al: The mouse homologue of the human acidic ribo somal phosphoprotein PO: A highly conserved polypeptide that is under translational control. Nucleic Acids Res 1989; 15:6408. 18 Makrides S, Chitpatima ST, Bandyopadhyay R, et al: Nucleotide sequence for a major messenger RNA for a 40 kilodalton polypeptide that is under translational control in mouse tumor cells. Nu cleic Acids Res 1988; 16:2349. 19 Chitpatima ST, Makrides S, Bandyopadhyay R, et al: Nucleotide sequence of a major messenger RNA for a 21 kilodalton polypeptide that is under translational control in mouse tumor cells. Nu cleic Acids Res 1988; 16:2350. 20 Schmid HP, Akhayat O, Martins DeSa C, et al: The prosome: an ubiquitous morphologically dis tinct RNP particle associated with repressed
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37 Rhoads RE: Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biochem Sci 1988;13:52-56. 38 Hariharan N, Perry RP: Functional dissection of a mouse ribosomal protein promoter: Significance of the polypyrimidine initiator and an element in the TATA-box region. Proc Natl Acad Sci USA 1990;87:1526-1530. 39 Hariharan N, Kelley DE, Perry RP: Equipotent mouse ribosomal protein promoters have a simi lar architecture that includes internal sequence elements. Genes Dev 1989;3:1789-1800.
Robert P. Perry Institute for Cancer Research Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia, PA 19111 (USA)
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32 Klausner RD, Harford JB: Cis-Trans models for post-transcriptional gene regulation. Science 1989;246:870-872. 33 Chitpatima ST, Brawerman G: Shifts in configu ration of the 5'-noncoding region of a mouse mes senger RNA under translational control. J Biol Chem 1988;263:7164-7169. 34 Duncan R, Milbum SC, Hershey JWB: Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in transla tional control. J Biol Chem 1987;262:380-388. 35 Walden WE, Godefroy-Colbum T, Thach RE: The role of mRNA competition in regulating translation. J Biol Chem 1981;256:11739— 11746. 36 Sonenberg N: Cap-binding proteins of eukaryotic messenger RNA: Functions in initiation and con trol of translation. Prog Nucleic Acid Res Mol Biol 1988;35:173-207.
Enzyme 1990;44:83-92
© 1990 S Karger AG, Basel 0013-9432/90/0444-0083S2.75/0
Translational Control of Ribosomal Protein Production in Mammalian Cells Robert P. Perry3, OdedMeyuhasb aInstitute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa., USA, and b Department of Developmental Biochemistry, Institute of Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem, Israel
Key Words. 5'-Un translated region • Ribosomal proteins ■ Polyribosomes • 5'-01igopyrimidine tract • Glucocorticoids Abstract. Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their under-representation in polyribosomes of normally growing cells. Recent studies have local ized the translational regulatory element to the 5' end of the rp mRNA and have demon strated that an oligopyrimidine tract, which adjoins the cap structure in all known vertebrate rp mRNAs, is an essential part of this element. Possible factors that might interact with the oligopyrimidine tract are discussed.
mechanism. Evidence for translational con trol of rp production has been observed dur ing developmental transitions of Dictyostelium discoideum [4], Drosophila melanogaster [5], Xenopus laevis [Amaldi and Pierandrei-Amaldi, this volume], and mouse myo blasts [6]. Selective regulation of rp mRNA translation also occurs when mammalian cells undergo a shift between growing and nongrowing states and during cellular re sponses to certain hormones and other stim uli [7-15]. In the present article, we will Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
The formation of eukaryotic ribosomes involves the coordinate production of four RNA components and more than 70 differ ent proteins. The synthesis of these ribo somal constituents is regulated in response to changes in physiological and develop mental states by a variety of mechanisms acting at all levels of gene expression from transcription to protein turnover [1-3]. In higher eukaryotes, the controlled translation of the mRNAs encoding ribosomal proteins (rp) is a particularly important regulatory
84
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General Features of rp mRNA Translation
The translational control of mammalian rp mRNAs was demonstrated by studies of their relative apportionment between trans lationally repressed mRNP particles and translationally active polyribosomes under various physiological and developmental conditions. In resting mouse fibroblasts, only about 20-30% of the rp mRNA is in a translationally active state, whereas in se rum-stimulated growing fibroblasts, this pro portion increases to about 65% [7, 9], Simi larly, in glucocorticoid-treated PI798 lym phosarcoma cells, which are arrested in growth and no longer produce ribosomes, the proportion of rp mRNA in polyribo somes is about 25 %, compared to about 65 % for exponentially growing cells [8, 10, 15]. In these latter studies, the apportionment be tween mRNP and polyribosomes was exam ined for 8 different rp mRNAs and various other mRNAs that encode general house keeping functions. Although the glucocorti coid treatment resulted in a marked decrease in the ribosome loading of 7 of the 8 rp mRNAs, the translation of the non-rp mRNAs was not significantly affected. Typi cal examples of such data are shown in fig ure 1. Clearly, there is some selectivity in the translational control of rp mRNAs under these conditions. Still another example of selective translational control of rp mRNA was observed in differentiating mouse myo blasts [6, 13]. In these cells, the proportion of
translationally active rp mRNA decreases from 45 to about 30% when myoblasts fuse into myotubes and increases to about 70% when myoblasts are treated with insu lin. In all of the studies cited above, it is clear that even when ribosomes are being synthe sized at maximum rates in rapidly proliferat ing cells, there is still a significant proportion of rp mRNA (about 30-40%) that is in a translationally repressed state. In contrast, only a minor fraction (< 10%) of most nonrp mRNAs is translationally repressed under the same conditions. Interestingly, a charac terization of cDNA clones for three mRNA species that are roughly equally distributed between free mRNP and polyribosomes in both mouse sarcoma 180 ascites cells and mouse erythroleukemia cells [16], revealed that one of them was an rp mRNA [17-19]. Although the other two mRNAs encode nonribosomal proteins, their extreme 5'ends bear a striking similarity to the rp mRNAs (see below). Another important feature of rp mRNA translation was deduced from a consider ation of the number of ribosomes associated with the translationally active fraction of rp mRNA in growing cells [8], As seen in the example of figure 1, the profiles of rp mRNAs in growing lymphosarcoma cells harvested 24 h after withdrawal of the hor mone are distinctly bimodal, suggesting two discrete populations of mRNA molecules which are distinguished by being either translationally inactive (free mRNP) or effi ciency translated (in polysomes containing 4-7 ribosomes). Considering the sizes of the rp mRNAs, this extent of ribosome loading indicates an average spacing of 31 codons per ribosome, which is similar to that of the non-rp mRNAs examined and believed to be Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
summarize the main features of translational control of rp mRNA in mammalian cells and try to provide some insight into the mecha nism^) responsible for this control.
Fig. 1. Translational control of rp mRNAs in PI798 lymphosarcoma cells. Cytoplasmic extracts of cells treated for 24 h with 10~7 M dexamethasone and either harvested immediately (Dexamethasone) or af ter incubation for an additional 24 h in the absence of hormone (Withdrawal) were fractionated on 15-45% sucrose gradients. RNA was extracted from each frac tion and aliquots representing equal volumes were dot blotted onto Nytran sheets. The blots were hy bridized with 32P-labelled probes specific for various rp mRNAs and autoradiographed. After melting off and decay of the rp signals, the blots were rehybrid ized with probes for selected nonribosomal mRNAs [actin, superoxide dismutase (SOD), glyceraldehyde-
85
3-phosphate dehydrogenase (GA3PD) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)] and autoradiographed again. Top two panels show the Ai60 profiles with the positions of the polyribosomes, monoribosomes (M) and 60S and 40S subunits indi cated. The other panels show the distributions of each mRNA species plotted as a percentage of the total of that species on the gradient. Open and solid circles denote rp mRNAs and non-rp mRNAs, respectively. The vertical dashed lines separate the polyribosome and subpolysome (free mRNP) fractions. Profiles from ‘withdrawal’ cells are indistinguishable from those of untreated cells [8].
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Table 1. Context of the AUG initiation codon in mammalian ribosomal protein mRNAs
C A G U Consensus rp General
-9
-8
-7
-6
-5
-4
-3
-2
-1
8 2
13 2 5 3
16 2
4 4 13 2
11
15 6 1
0 13 10 0
8 10 3 2
7 4 9 3
5 N G
C C
4 C C
G G
0 10 2 G C
C
1
c C
A G A G
A C
N
c
c
A
U
G
+4 3 6 13
A A
U____G U G
G G
The numbers in the table represent the frequency of occurrence of each nucleotide at the indicated position in a survey of 23 mammalian ribosomal protein mRNAs listed in Genbank. The tabulated sequences were as follows: h/mPo, hPl, rS8, rSlO, rS12, hS14, mS16, hS17, rS26, rL5, rL6, mL7, mL7a, rL18, rL19, rL21, rL26, mL30, rL31, mL32, rL34, rL36a and rL37. h, m and r signify human, mouse and rat proteins; S and L designate small and large subunit proteins, respectively.
changes in the content or activity of these ligands or of the general translation factors that compete with them.
Identification of the Translational Regulatory Element in rp mRNAs
The selective nature of the translational control of rp mRNAs suggests that they have some distinctive property that is recognized by the translational apparatus and/or by pro teins of the mRNP particles. A survey of the sequences spanning the AUG initiation co don in mammalian rp mRNAs (table 1 ) does not reveal any consistent pattern or trend that might be associated with an impairment of initiation efficiency. Indeed, the rp mRNAs examined to date conform to the general consensus and all contain a purine at the — 3 position, a feature that is believed to be especially important for efficient transla tion [21]. Although inverted repeat se quences capable of intrastrand base pairing Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
close to the theoretical maximum for any mRNA. Such bimodal distributions, which have been observed in other types of mam malian cells [6, 16] and in Xenopus embryos [Amaldi and Pierandrei-Amaldi, this vol ume], indicate that the rp mRNAs alternate between a repressed and an active state, and that, when in the active state, they are trans lated at near maximum efficiency. To account for the observed translational behavior of rp mRNAs, one may consider mechanistic models based on selective pack aging of rp mRNA into RNP particles [20] or the association of rp mRNA with a specific factor that limits its access to ribosomes and other components required for translation. Such ligands would presumably bind to the rp mRNAs with relatively high affinity and preempt interactions with translational fac tors. In the absence of ligands, the rp mRNAs are evidently capable of high effi ciency translation. The relative proportions of rp mRNA in translationally active and repressed states could be regulated by
Translational Control of Ribosomal Protein Production
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Table 2. 5' oligopyrimidine tracts in selected mammalian rp mRNAs rp mRNA
5' terminal sequence
mS16
CCUUUUCCG
mL32
CUUCUUCCUCG
mL30
CCUUUCUCG
mL7
CUCUCUUCUUUUCCG
Number of consecutive pyrimidines
References
8
23
10
24
8
25
14
26
*
mL7a
CUUUCUUUCUCCA
8/12
rL35a
CUCUUUCU
8
28
hS14
CUCUUUCCG
8
29
hS17
CCUCUUUUA
8
30
27
+
h, m and r refer to human, mouse and rat mRNAs, respectively. Two start sites detected. The center of a diffuse starting region.
are found in the 5'untranslated regions (UTRs) of some rp mRNAs, such repeats are not prevalent among all rp mRNAs. More over, the 5'UTRs of rp mRNAs are generally relatively short (25-50 nucleotides) and only one (L5) has been found to contain an AUG upstream of the true initiator codon [22], One common feature noted in all se quenced vertebrate rp mRNAs is an oligopy rimidine tract at the 5' terminus. In cases for which complete sequence information is available, this element is found to consist of a C residue at the cap site followed by an uninterrupted sequence of 7-13 pyrimidines (table 2). The fact that some non-rp mRNAs that exhibit the same translational control properties as rp mRNA also have a 5' termi nal oligopyrimidine tract [18, 19] suggests that this sequence may be a critical part of the translational regulatory element (TLRE).
Experiments designed to verify this con jecture and to localize the TLRE within the rp mRNAs have recently been carried out with PI798 lymphosarcoma cells, which, as described above, exhibit dramatic control of rp mRNA translation when exposed to the glucocorticoid hormone, dexamethasone [31]. In these experiments, the cells were transfected with chimeric genes capable of producing mRNAs in which rp and non-rp coding regions were linked in various combi nations to the 5'UTR of an authentic or mutated rp mRNA or to the 5'UTR of ßactin mRNA, a typical non-rp mRNA that is not translationally controlled (fig. 2). The apportionment of the chimeric mRNAs in polyribosomes and in free mRNP of hor mone-treated and control cells was deter mined and compared to that of an endoge nous actin and rp mRNA. The typical de crease in ribosomal loading of rp L32 mRNA Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
* +
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El ID •
pAct-L32 +74
-370 pAct-GH (74 n)
+167
266 pL30-GH (61 n)
l
D -179
+29
pS16-GH (29 n)
a +1
S16 wild-type
CCTTTTCC
pS16CM5-GH (27 n)
GAGTGACC I
pS16CM3-GH (27 n)
ACTTTTCC
Act-l
Act-L32 100-1
Ì-GH
S16CM5-GH 100
100
S16CM3-GH 100
80-
80 -
80
80-
60-
60 -
60-
60-
40-
40
40-
40
20-
20-
20-
CO
CD
E CO
>>
R
c
1
0 Con Dex
0
Con
Dex
0 Con Dex
o
b Con Dex
gradients and then analyzed as described in figure 1 for mRNAs produced by the transfected chimeric genes (hatched bars), the endogenous ß-actin gene (solid bars) and the endogenous rpL5 gene (open bars). The height of each bar represents the percentage of each mRNA that is in the polyribosome fraction.
in dexamethasone-treated cells was abol ished when its 5' end was replaced with that of ß-actin. Moreover, growth hormone (GH) mRNA acquired the translational control properties of an rp mRNA when its 5' end was replaced by that of rpL30 or rpS16, but
not when it was replaced by that of ß-actin. These results indicate that the 5' end of the rp mRNAs is necessary and sufficient to account for their translational control prop erties. Similar experiments, carried out with mouse myoblasts [Lewis Bowman, personal Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
Fig. 2. Localization of the translational control element in rp mRNA. a Schematic representation of the chimeric genes that were transfected into PI798 cells, b Cytoplasmic extracts of transfected cells, either untreated (Con) or exposed for 24 h to 10~7 M dexamethasone (Dex), were fractionated on sucrose
communication] or with X. laevis embryos [Amaldi and Pierandrei-Amaldi, this vol ume], have led to essentially the same con clusion. It is interesting to note that as few as 29 5'-terminal nucleotides of rpS16 are suffi cient to confer translational control on GH mRNA, thus localizing the TLRE to a rela tively short segment with no significant base pairing potential. Translational control was abolished when the chimeric genes were made with rpS16 mutants that cause the 5'-oligopyrimidine tract to be interrupted with purines or to be shortened by a couple of nucleotides. The loss of translational repression was demon strated by monitoring the polyribosomal as sociation of the mutant chimeric mRNAs (fig. 2), as well as by a direct immunoassay of the rate of synthesis of hGH [31]. These results indicate that the 5'-oligopyrimidine tract is a critical part of the TLRE and that either the terminal C residue, a minimal length of 7 or 8 consecutive pyrimidines or both of these features are essential for its activity. All of the rp 5'-terminal sequences known to date are consistent with this con clusion (table 2). There may also be impor tant structural information in the sequences immediately downstream of the pyrimidine tract. Although there are no obvious com mon sequence motifs in this region, there could conceivably be subtle patterns of nu cleotides that contribute importantly to the overall structure of the extreme 5' end.
Possible Trans-Acting Factors that Interact with the TLRE
Beyond a precise definition of the cis-act ing TLRE, it is also important to identify the trans-acting factors that determine the activ
89
ity of this regulatory element. Conceivably, there might be a factor that specifically binds to the TLRE and prevents the mRNA from interacting with ribosomes and other com ponents that are essential for protein synthe sis. An example of such a factor is the pro tein that represses the translation of ferritin mRNA by binding to a stem-loop structure in the 5TJTR of this mRNA [32], If an anal ogous TLRE-specific factor exists, it would presumably be quantitatively regulated or qualitatively modified in response to changes in cellular growth rate. An interac tion of protein(s) with the TLRE of mRNA that is sequestered in translationally inactive mRNP particles was suggested by a study of mouse P21 mRNA, a non-rp mRNA which has a 5'-terminal oligopyrimidine tract and which is subject to growth-dependent trans lational control [33]. Treatment of P21 mRNA with RNase T1 revealed that the G residues immediately downstream of the 5' pyrimidines are much more accessible when the mRNA is engaged in polyribosomes than when packaged in mRNP particles. Addi tional evidence for the possible involvement of such mRNP proteins in translational con trol has been discussed elsewhere [20]. In addition to a TLRE-specific factor, translational control may involve a factor that is part of the general protein-synthesiz ing machinery. Because the rp mRNAs nor mally exist in a delicate balance between translationally active and inactive states, they should be especially sensitive to changes in the content or activity of a critical translational initiation factor. If such a fac tor had a particularly low affinity for rp mRNA, decrease in its activity or content could lead to a selective diminution of rp mRNA translation. The fact that dexamethasone reduces the abundance of the mRNAs Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
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scription and turnover of the various rp mRNAs [7, 39], helps insure a well-balanced production of ribosomal proteins under a variety of physiological and developmental conditions.
References 1 Meyuhas O: Ribosomal protein gene expression in proliferating and nonproliferating cells; in Stein GS, Stein JL (eds): Recombinant DNA and Cell Proliferation. Academic Press, Orlando, 1984, pp 243-271. 2 Jacobs-Lorena M, Fried HM: Translational regu lation of ribosomal protein gene expression in eukaryotes; in Ilan J (ed): Translational Regula tion of Gene Expression, Plenum Press, New York, 1987, pp 63-85. 3 Mager WH: Control of ribosomal protein gene expression. Biochim Biophys Acta 1988;949:1 — 15. 4 Steel LF, Jacobson A: Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development. Mol Cell Biol 1987;7:965-972. 5 Al-Atia GR, Fruscolom P, Jacobs-Lorena M: Translational regulation of mRNAs for ribosomal proteins during early Drosophila development. Biochemistry 1985;24:5798-5803. 6 Agrawal MG, Bowman LH: Transcriptional and translational regulation of ribosomal protein for mation during mouse myoblast differentiation. J Biol Chem 1987;262:4868-4875. 7 Geyer PK, Meyuhas O, Perry RP, et al: Regula tion of ribosomal protein mRNA content and translation in growth-stimulated mouse fibro blasts. Mol Cell Biol 1982;2:685-693. 8 Meyuhas O, Thompson EA, Perry RP: Glucocor ticoids selectively inhibit translation of ribosomal protein mRNAs in PI798 lymphosarcoma cells. Mol Cell Biol 1987;7:2691-2699. 9 Kaspar RL, Rychlik W, White MW, et al: Simul taneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of euka ryotic initiation factor 4E after mitogenic stimula tion of Swiss 3T3 cells. J Biol Chem 1990;265: 3619-3622. Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
encoding the translational initiation factors eIF-4A, eIF-2a and eIF-4D by 60-70% in PI798 cells [15] is consistent with this idea. Moreover, an increase in the translational efficiency of rp mRNAs during transition of Swiss 3T3 cells from nongrowing to growing states occurs simultaneously with enhanced phosphorylation of the initiation factor elF4E [9], This factor, which is also known as the cap-binding protein, constitutes, to gether with eIF-4A and a 220-kD protein, the eIF-4F complex. Due to the relatively low abundance of eIF-4E [34], the entire elF4F complex is a limiting component in the binding of eukaryotic mRNAs to the ribo some. Since this step is generally considered to be the overall limiting step in translation [35], eIF-4F is a prime candidate for modu lating translation efficiency. Indeed, the lat ter has been implicated in discrimination between weak and strong mRNAs [for re view see Sonenberg, 36, and Rhoads, 37], Since rp mRNAs apparently have normal cap structures, as inferred from the heteroge nous products observed in SI nuclease pro tection analyses [23, 38], they should be able to bind eIF-4F. Thus, the novel 5'-terminal structure of the TLRE could define a binding site for specific inhibitory ligands and also help determine the ability of these ligands to be displaced by translational initiation factors. Irrespective of the exact mechanism, it would appear that the oligopyrimidine tract endows rp mRNAs and other mRNAs bear ing a similar 5' terminus with the property of being underutilized for translation, even un der optimal growth conditions, and of being exquisitely sensitive to conditions that ac company changes in growth rate. This trans lational control property, together with the remarkable equivalence in the rates of tran-
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21 22
23
24
25
26
27
28
29
30
31
mRNPs and containing specific ScRNA and a characteristic set of proteins. EMBO J 1984;3:29— 34. Kozak M: The scanning model for translation: An update. J Cell Biol 1989;108:229-241. Tamura S, Kuwano Y, Nakayama T, et al: Molec ular cloning and nucleotide sequence of cDNA specific for rat ribosomal protein L5. Eur J Biochem 1987;168:83-87. Wagner M, Perry RP: Characterization of the multigene family encoding the mouse SI6 ribo somal protein: strategy for distinguishing an ex pressed gene from its processed pseudogene coun terparts by an analysis of total genomic DNA. Mol Cell Biol 1985;5:3560-3576. Dudov KP, Perry RP: The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell 1984;37:457— 468. Wiedemann LM, Perry RP: Characterization of the expressed gene and several processed pseudo genes for the mouse ribosomal protein L30 gene family. Mol Cell Biol 1984;4:2518-2528. Meyuhas O, Klein A: The mouse ribosomal pro tein L7 gene: Its primary structure and functional analysis of the promoter region. J Biol Chem 1990;265:11465-11473. Huxley C, Williams T, Fried M: The mouse rpL7a gene is typical of other ribosomal protein genes in its 5' region but differs in being located in a tight cluster of CpG-rich islands. Nucleic Acids Res 1990;18:5353-5357. Kuzumaki T, Tanaka T, Ishikawa K, et al: Rat ribosomal protein L35a multigene family: molec ular structure and characterization of three L35arelated pseudogenes. Biochim Biophys Acta 1987; 909:99-106. Rhoads DD, Dixit A, Roufa DJ: Primary struc ture of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 1986;6:2774— 2783. Chen I-T, Roufa DJ: The transcriptionally active human ribosomal protein S17 gene. Gene 1988; 70:107-116. Levy S, Avni D, Hariharan N, et al: The oligopyrimidine tract at the 5' end of mammalian ribo somal protein mRNAs is required for their trans lational control. Proc Natl Acad Sci USA 1991,88: 3319-3323. Downloaded by: University of Exeter 144.173.6.94 - 6/6/2020 6:54:59 AM
10 Meyuhas O, Baldin V, Bouche G, et al: Glucocor ticoids repress ribosome biosynthesis in lympho sarcoma cells by affecting gene expression at the level of transcription, posttranscription and trans lation. Biochim Biophys Acta 1990;1049:38-44. 11 DePhilip RM, Rudert WA, Lieberman I: Prefer ential stimulation of ribosomal protein synthesis by insulin and in the absence of ribosomal and messenger ribonucleic acid formation. Biochemis try 1980;19:1662-1669. 12 Ignotz GG, Hokari S, DePhilip RM, et al: Lodish model and regulation of ribosomal protein synthe sis by insulin-deficient chick embryo fibroblasts. Biochemistry 1981;20:2550-2558. 13 Hammond ML, Bowman LH: Insulin stimulates the translation of ribosomal proteins and the tran scription of rDNA in mouse myoblasts. J Biol Chem 1988;263:17785-17791. 14 Schmidt T, Chen PS, Pellegrini M: The induction of ribosome biosynthesis in a nonmitotic secre tory tissue. J Biol Chem 1985;260:7645-7650. 15 Huang S, Hershey JWB: Translational initiation factor expression and ribosomal protein gene ex pression are repressed coordinately but by differ ent mechanisms in murine lymphosarcoma cells treated with glucocorticoids. Mol Cell Biol 1989; 9:3679-3684. 16 Yenofsky R, Cereghini S, Krowczynska A, et al: Regulation of mRNA utilization in mouse erythroleukemia cells induced to differentiate by expo sure to dimethyl sulfoxide. Mol Cell Biol 1983;3: 1197-1203. 17 Krowczynska AM, Coutts M, Makrides S, et al: The mouse homologue of the human acidic ribo somal phosphoprotein PO: A highly conserved polypeptide that is under translational control. Nucleic Acids Res 1989; 15:6408. 18 Makrides S, Chitpatima ST, Bandyopadhyay R, et al: Nucleotide sequence for a major messenger RNA for a 40 kilodalton polypeptide that is under translational control in mouse tumor cells. Nu cleic Acids Res 1988; 16:2349. 19 Chitpatima ST, Makrides S, Bandyopadhyay R, et al: Nucleotide sequence of a major messenger RNA for a 21 kilodalton polypeptide that is under translational control in mouse tumor cells. Nu cleic Acids Res 1988; 16:2350. 20 Schmid HP, Akhayat O, Martins DeSa C, et al: The prosome: an ubiquitous morphologically dis tinct RNP particle associated with repressed
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37 Rhoads RE: Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biochem Sci 1988;13:52-56. 38 Hariharan N, Perry RP: Functional dissection of a mouse ribosomal protein promoter: Significance of the polypyrimidine initiator and an element in the TATA-box region. Proc Natl Acad Sci USA 1990;87:1526-1530. 39 Hariharan N, Kelley DE, Perry RP: Equipotent mouse ribosomal protein promoters have a simi lar architecture that includes internal sequence elements. Genes Dev 1989;3:1789-1800.
Robert P. Perry Institute for Cancer Research Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia, PA 19111 (USA)
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32 Klausner RD, Harford JB: Cis-Trans models for post-transcriptional gene regulation. Science 1989;246:870-872. 33 Chitpatima ST, Brawerman G: Shifts in configu ration of the 5'-noncoding region of a mouse mes senger RNA under translational control. J Biol Chem 1988;263:7164-7169. 34 Duncan R, Milbum SC, Hershey JWB: Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in transla tional control. J Biol Chem 1987;262:380-388. 35 Walden WE, Godefroy-Colbum T, Thach RE: The role of mRNA competition in regulating translation. J Biol Chem 1981;256:11739— 11746. 36 Sonenberg N: Cap-binding proteins of eukaryotic messenger RNA: Functions in initiation and con trol of translation. Prog Nucleic Acid Res Mol Biol 1988;35:173-207.
