Role of cytoplasmic mRNP proteins in translation

Biochimie (1992) 74, 477--483 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Pads

477

Role of cytoplasmic mRNP proteins in translation WB Minich, LP Ovchinnikov Institute of Protein Research, Russian Academy of Sciences, 142292, Pushchino, Moscow Region, Russia

(Received 26 August 1991; accepted 15 November 1991)

Summary m Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the lepressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per tool of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA - 2:1, mol/mol), this protein promotes the translation process. In free mRNPs (p50:mRNA = 4: I, tool/tool) this protein is a translational repressor, a component of the above mentioned mRNA repressor/activator regulatory system. retieulocyte / translation / mRNA / mRNPs / mRNP proteins Introduction

In 1964 Spirin and coworkers, studying fish embryo cytoplasmic extracts by the method of sucrose gradient centrifugation, noted that newly synthesized ~4Clabelled cytoplasmic R N A (mRNA) sedimented immediately after the 80S ribosomes from 20S to 75S. Attention was drawn to the fact that similarly sedimenting labelled components were also observed in the case when the embryos were preincubated with 14C-labelled amino acids. The coincidence of label distribution profiles in both cases, as well as the high sedimentation coefficients of the postribosomal labelled components suggested that this could be some kind of newly formed mRNA-protein complexes [ 1, 2]. This assumption was confirmed by CsCI density gradient centrifugation of formaldehyde fixed labelled 20-75S material. The density distribution of the nucleic acid label and that of the protein coincided, indicating that both pertain to the same type of particles. The buoyant density of these particles was approximately 1.40 g/cm 3, corresponding to the protein:RNA weight ratio of 4:1. These mRNA-cmaving

ribonucleoprotein particles with a relatively low characteristic buoyant density, were called informosomes [2, 3]. It was proposed that informosomes are a form of existence of masked mRNA, and that the informosome-forming protein could be considered as a repressor of translation [4]. Subsequent studies showed that informosomes are a universal form of existence of all mRNA and its precursors in eukaryotic cell (reviewed in [5-7]). Viral m R N A was also found in the eukaryotic cells within mRNPs with properties of informosomes ([8], rewieved in [5-7]). It is generally accepted now that eukaryotic mRNA, at all stages of its lifetime, is present in the form of ribonucleoprotein particles and, consequently, always binds to certain proteins. Informosomes can be classified, depending on their intracellular location, as nuclear (hnRNPs) and cytoplasmic ones. Cytoplasmic mRNPs, in turn, can exist in a free form (free mRNPs) and as a part of polyribosomes (polyribosomal mRNPs) [5-7, 9-12]. The three classes of informosomes are different in their protein composition, hnRNPs contain up to a hundred of proteins. Among them, the protein family

478 with molecular masses of 34-42 kDa (informatins) predominates (reviewed in [6, 13, 14]). Free cytoplasmic mRNPs contain about a dozen major proteins with molecular masses from 150 to 30 kDa. Polyribosomal mRNPs contain two major proteL-.s with molecular masses of 70 kDa and 50 kDa, as well as some minor polypeptides (reviewed in [6, 7, 15, 16]). A cytoplasmic mRNP protein with a molecular mass of about 70 kDa, associated with poly(A)-tails of mRNAs, is the most characterized at present. This protein is common for most, if not for all eukaryotes. Cloning and sequencing of genes of poly(A)-binding protein of yeast, human and Xenopus has revealed that this protein is highly conservative in evolution [17-19]. In 1978, Spirin proposed that mRNA in the eukaryotic cell carried on itself the proteins which are required for its own biogenesis, existence and functioning ('Omnia mea mecum porto') [20]. Proceeding from this hypothesis, the protein moiety of hnRNPs consists of proteins serving processing of mRNA and its transport from the nucleus to the cytoplasm. Free cytoplasmic mRNPs should contain translation repressors and proteins preventing mRNA degradation during its storage. Polyribosomal mRNP proteins should take part in the translation process.

There are a number of experimental data which are in good agreement with the 'Omnia mea mecum porto" hypothesis. It was shown that hnRNP preparations contain two poly(A)-synthetase activities, enzymes for mRNA capping, endonucleases, protein kinase and protein phosphatase activity. A set of low-molecularweight RNPs (snRNPs) taking part in the process of mRNA splicing, has been also found wi~hh'~ ~a'~,~,,rPs (reviewed in [7, 13, 14]). It has been shown that cytoplasmic mRNP proteins can t~.~ke part in control of mRNA stability [21, 22], in compartmentalization of mRNA in the cytoplasm [23], in mRNA masking and in regulation of mRNA translation [22, 24-28]. There are data also on the presence of some initiation factors within free [29] and polyribosomal [30, 31] mRNPs. Genetic and biochemical studies have shown the crucial role of 70 kDa poly(A)-binding protein in cell viability [32] and have demonstrated its participation in initiation of translation [33-35]. In this report we present the results of our studies with free and polyribosomal mRNPs from rabbit reticulocytes. Translation of mRNPs in cell-free systems of protein biosynthesis was investigated. Data on the role of cytoplasmic mRNP proteins (in particular a major 50 kDa protein) in protein biosynthesis were analyzed.

Isolation and properties of cytoplasmic mRNPs from rabbit reticulocytes

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Fig 1. SDS gel electrophoresis of polyribosomal mRNP proteins (a), free mRNP proteins (b), high salt treated core free mRNP proteins (c), isolated protein p50 (d).

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated by chromatography on oligo(dT)cellulose [36, 37]. Polyribosomes were dissociated by EDTA. Binding of mRNPs with oligo(dT)-cellulose was performed at a moderate ionic strength - 150 mM NaCl - at 4°C. Bound mRNPs were eluted in NaClfree buffer at 37°C. RNA electrophoresis and analysis of translation products have shown that both polyribosomal and free mRNPs contain mainly globin 9S mRNA. Polyribosomal mRNPs have a sedimentation coefficient of about 13S and a buoyant density in CsCl of about 1.45 g/cm3, corresponding to the protein:RNA weight ratio of 2:1. Free mRNPs have a sedimentation coefficient of about 20S and a buoyant density in CsCI of about 1.40 g/cm3, corresponding to the protein:RNA weight ratio of 4:1 [37, 38]. Polyribosomal mRNPs contain two major proteins with molecular masses of 70 kDa (poly(A)-binding protein) and 50 kDa, as well as some minor components (fig l a). Free mRNPs contain several major proteins from 150 to 30 kDa (fig lb). Protein 50 kDa predominates comprising, according to gel scanning experiments, 2030% of the total mass of mRN'P proteins. The results obtained are in good agreement with data available in literature (see Introduction).

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mRNA or mRNA-component of mRNP, ).tg/ml Fig 2. Translation of polyribosomal mRNPs (a, d), free mRNPs (b, e) and core free mRNPs, containing predominantly p50 (c, f) in cell-free systems of protein synthesis from rabbit reticulocytes (a, b, e) and wheat germs (d, e, f). (= =), mRNPs; (o o), mRNA, isolated from the corresponding classes of mRNPs. The rabbit reticulocyte cell-free system treated with micrococcal nuclease and wheat germ cell-free system were prepared as in [47, 48], respectively. An increase of ionic strength in the process of free mRNP isolation (at the stage of binding of mRNPs with oligo(dT)-ceUulose) from 150 to 500 mM NaCl leads to isolation of a complex of mRNA with the predominating 50 kDa protein (pS0) (fig lc). In these core free mRNPs pS0 comprises, according to gel scanning experiments, approximately 70% of the total protein. These mRNPs have a buoyant density in CsCl of about 1.65 g/cm3, corresponding to the protein:RNA weight ratio of 0.5:1.

Translation of mRNPs in rabbit reticulocyte lysate Translation of polyribosomal and free mRNPs fiam rabbit reticulocytes was compared with that of mRNA

isolated from the same particles. It is shown (fig 2a,b) that mRNA within polyribosomal and free mRNPs, as welt as deproteinized mRNA, are active in translation in the rabbit reticulocyte lysate. Some differences in translation of mRNPs and mRNA are worth mentioning: 1) the maximal efficiency of mRNP translation is 20-30% higher than that of mRNA; 2) at low conceutrations free mRNPs, but not polyribosomal ones, are translated less efficiently than deproteinized mRNA. This result indicates that some part of free mRNPs can exist in a translationally repressed state; 3) a high concentration of mRNA in the form of mRNP does not inhibit protein biosynthesis, whereas :he same concentration of deproteinized mRNA strongly inhibits this process.

480 Inhibition of protein biosynthesis by an excess mRNA [39, 40] as well as high concentrations of rRNA [41 ] and polynucleotides [42, 43] have been described previously. It has been shown that RNA and polynucleotides inhibit the process of translation at the step of initiation. It has been suggested that these compounds inhibit protein biosynthesis by interacting with some initiation factors [39-43], in particular with eIF-2 [40]. Our results show that mRNA inhibits protein biosynthesis much more effectively than rRNA from E coli [37]. This suggests a specific binding between the mRNA and the translation factors which limit protein biosynthesis under excess templates, mRNPs proved to be unable to overcome the inhibition of the cell-free system caused by an excess mRNA [37]. This indicates that mRNPs did not contain any detectable amount of the translation factors limiting protein biosynthesis under an excess mRNA. On the grounds of these results we believe that cytoplasmic mRNP proteins play a specific role in the process of translation. These proteins can organize the mRNA structure shielding it from occasional interactions with the translation factors and/or increase the cooperativity of interaction of the factors with mRNA (fig 3). This may result in a more effective usage of the translation factors in protein biosynthesis. The proposed function of mRNP proteins can be of essential significance in protein biosynthesis. Data have been reported that in reticulocytes mRNA is present in an excess amount and that protein biosynthesis in reticu!ocyte iysates is limited by initiation factors ([44] reviewed in [45]). Translation of mRNPs in a wheat germ cell-free system

Translation of polyribosomal and free mRNPs from rabbit reticulocytes and that of mRNA isolated from

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A Fig 3. The hypothetical scheme of interaction of initiation factors with mRNA and mRNPs under an excess of templates in a cell-free system.

these particles in a wheat germ cell-free system is shown in figure 2d, e. Polyribosomal mRNPs, as well as mRNA isolated from polyribosomal and free mRNPs, are effectively translated in this system. At the same time, the translation of free mRNPs in a wheat germ cell-free system proceeds far less efficiently (a 10--25% level of mRNA translation) (fig 2e). Analysis of polyribosome formation has shown that translation of free mRNPs in a wheat germ cell-free system is inhibited at the initiation step [38]. These results indicate that free mRNPs from rabbit reticulocytes contain a component preventing the initiation of free mRNP translation in a wheat germ cellfree system. This component is absent in preparations of polyribosomal mRNPs and deproteinized mRNA. Translation of free mRNPs from rabbit reticulocytes in a wheat germ cell-free system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes [38]. Proceeding from these data we suggest the existence of a special repressor/activator system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. The activator of translation of rabbit reticulocyte free mRNPs is absent in wheat germs.

Translation of the core free mRNPs in cell-free systems

Figure 2c, f shows that high salt treated free mRNPs, containing predominantly p50 (core free mRNPs), nevertheless retain all translational features typical for native free mRNPs: l) the maximal efficiency of core free mRNPs translation in reticuloeyte lysates is higher than that of deproteinized mRNA; 2) high concentrations of mRNA in the form of core free mRNPs inhibited translation in rabbit reticulocyte cell-free system to a lesser extent th-~.n the same amounts of deproteinized mRNA; 3) at low concentrations core free mRNPs are translated in reticulocyte lysates less efficiently than deproteinized mRNA; 4) core free mRNPs are poorly translated in a wheat germ cell-free system (20-40% of the level of mRNA translation). We believe, based on these results, that it is p50 that is responsible for the above mentioned features of free mRNPs translation in the two cell-free systems. We believe that: i) p50 reverses the inhibitory effect of an excess mRNA on protein biosynthesis; and ii) p50 is a repressor which prevents mRNA translation in the cell-free system.

481 Table I. Some properties of protein p50. Molecular mass

= 50 kDa

lsoelectric point

= 9.5

Binding constant with globin mRNA

= 2.5 10s M-I (100 mM KAc, 4°C) (interacts with = 20 nucleotides)

Relative affinity

poly G > poly U > globin mRNA = 16S rRNA > poly A > poly C

Content (mol p50 per mol of globin mRNA

= 2 in polyribosomal mRNPs, = 4 in free mRNPs

Modifications

phosphorylation

of mRNA (results not shown). This result suggests that pS0 inhibits protein biosynthesis by binding with mRNA and by preventing its normal interaction with some components of the translation apparatus. Thus pS0 can play a dual role in the mRNA translation, stimulate the translation at a low p50:mRNA ratio and inhibit it at a higher ratio. Basing on these results and on the fact that the content of pS0 per mol of mRNA in free mRNPs is twice as high as in polyribosomal ones, we suggest that pS0 plays an opposite role in the two classes of mRNPs. In polyribosomal mRNPs

Isolation and some properties of protein p50 from rabbit reticulocyte mRNPs We have developed a method of isolating the major 50 kDa protein (pS0) from rabbit reticulocyte free mRNPs (fig ld) and investigated its features. Table I shows some properties of the pS0 protein ([46]; Minich, in preparation). pS0 is a basic protein, its isoelectric point being about 9.5. nitrocellulose filter analysis has shown that pS0 interacts with globin mRNA with an association constant of = 2.5 10s M-] (100 mM KAc, 4°C). One protein molecule binds = 20 nucleotides. Various polyribonucleotides have the following relative affinity to pS0: poly G > poly U > globin mRNA = 16S rRNA > poly A > poly C. Amino acid analysis and O'Farrell's two-dimensional gel electrophoresis have shown the indentity of 50 kDa proteins of free and polyribosomal mRNPs. However, according to gel scanning experiments, the content of this protein per tool of mRNA in free mRNPs is twice as much as in polyribosomal ones (-- 4 and = 2 mol of protein per mol of globin mRNA, respectively). The pS0 protein can be phosphorylated both in vitro and in vivo.

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Study of the effect of p50 on mRNA translation The p50 effect on the mRNA translation was tested in rabbit reticulocyte and wheat germ cell-free systems, containing high concentrations of globin mRNA (= 100 l.tg/ml). The used amounts of mRNA inhibited the systems up to = 20% of their maximal activity at optimal mRNA concentrations. An addition of p50 to such systems stimulated translation up to the level of optimal activity of these systems. A further increase of the p50:mRNA ratio in the cell-free systems caused inhibition of protein biosynthesis (fig 4). Inhibition of protein biosynthesis by pS0 was reversed by an excess

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p 5 0 / m R N A ratio,w/w Fig 4. Effect of p50 on globin mRNA translation in cellfree systems of protein synthesis from rabbit reticulocytes (a) and wheat germ (b). The rabbit reticulocyte cell-free system treated with micrococcal nuclease and wheat germ cell-free system were prepared as in [47, 48], respectively. Cell-free systems contained 100 lxg/ml of globin mRNA and different amounts of pS0,

482 ( p 5 0 : m R N A = 2:1, mol/mol), this protein promotes translation. In free m R N P s ( p 5 0 : m R N A = 4:1, mol/ mol), it functions as a translation repressor, a c o m p o nent of the above described repressor/activator system, which controls m R N A distribution between free m R N P s and p o l y r i b o s o m e s in rabbit reticulocyte. The more detailed analysis o f the curves in figure 4 shows that p50 inhibits protein biosynthesis in wheat germ cell-free system at a considerably smaller p 5 0 : m R N A ratio than in the cell-free s y s t e m from rabbit reticulocytes. This is in good a g r e e m e n t with the fact that free m R N P s are inactive in the wheat g e r m cell-free system but are active in the rabbit reticulocyte lysates. Thus the efficiency o f m R N A translation m a y depend on two parameters: the p50 to m R N A ratio and concentration of activator, associated with ribosomes of rabbit reticulocytes.

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