Eur. J. Biochem. 62. 509-517 (1976)

Molecular Modifications Associated with Ageing of Globin Messenger RNA in vivo Pierre NOKIN, Georges HUEZ, Gtrard MARBAIX, Arsene BURNY, and Hubert CHANTRENNE Laboratoire de Chimie Biologique, Departement de Biologie Molhlaire, Universite Libre de Bruxelles (Received July lX/October 28, 1975)

Using polyacrylamide gel elution-electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be fractionated into several populations of molecules differing by their mean poly(A) content. Both a and fl globin mRNA are heterogeneous with respect to their electrophoretic mobilities. With the conditions used no separation of a and fl globin mRNA occurs during electrophoresis. From the specificradioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3' end of globin mRNA molecules becomes shorter with ageing. This shortening occurs on a, as well as fl, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution-electrophoresis and elution of poly(U)-Sepharose-bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell-free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA ageing.

Most eukaryotic messenger RNAs contain a polyadenylate sequence covalently bound at the 3' end of the molecule [l]. It seems that this poly(A) stretch, added to the mRNA precursor by a post-transcriptional process in the nucleus, diminishes in size after its appearance in the cytoplasm [2- 41. Consequently, fractionation of a purified mRNA species based on the length of its polyadenylate sequence should make it possiblc t o separate mRNA populations of different ages. The comparison of these different mRNA fractions should help us to understand messenger RNA ageing. In this paper we describe a new technique of polyacrylamide gel elution-electrophoresis, which can resolve a highly purified globin messenger RNA preparation in several subfractions on the basis of the length of the poly(A) tract. Using highly purified 32P-labelledrabbit globin mRNA we show that both M and /j globin mRNA become shorter in vivo with ageing and that this decrease in size is due to the shortening of the poly(A) stretch. Moreover, we show that during messenger RNA ageing one or more further modifications occur, which decrease the ability of the mRNA to direct protein synthesis in vitro. Ahhreviurions. cDNA, single-stranded DNA complementary to an RNA molecule. Abbreviations for polynucleotides follow CBN. Recommendations; see Eur. J . Biochem. 15, 203-208 (1970). Enzyme. S, nuclease (EC 3.1.4.21).

MATERIALS AND METHODS

Solutions Used Phenol Chloroform Mixture. A mixture of 100 g phenol, 14 ml cresol and 0.5 g 8-hydroxyquinoline is equilibrated several times with buffer A until the correct pH 8.0 is reached in the aqueous phase. An equal volume of a chloroform isoamyl alcohol mixture (24/1) is then added. Bujytrs. Buffer A : 0.01 M Tris, 0.001 M EDTA, 0.15 M NaC1, 1 "/, sodium dodecylsulphate brought to pH 8.3 with HCl. Buffer B: 0.01 M Tris, 0.001 M EDTA, 0.2 sodium dodecylsulphate brought to pH 7.6 with HCl. Buffer C : 0.01 M Tris, 0.015 M KCI brought to pH 7.5 with HC1. cDNA hybridization buffer: 0.01 M Tris, 0.4 M NaCI, 0.001 M EDTA, 0.1 sodium dodecylsulphate brought to pH 7.6 with HC1. Nuclease S1 buffer ( l o x ) : 0.3 M acetic acid, 0.5 M NaC1, 0.01 M ZnSO, brought to pH 4.6 with NaOH. Nuclease S1 solution: a purified S, nuclease solution was prepared according to Vogt [5]. 0.1 pl of this enzyme solution completely degrades 15 pg of single-stranded DNA within 10 min at 45 "C in 0.25 ml of S, nuclease buffer.

(x

Globin mRNA Preparation Rabbit globin mRNA was isolated from the 15-S messenger ribonucleoprotein released from reticulo-

510

cyte polysomes by EDTA treatment and purified by sucrose gradient centrifugation [6,7]. In the experiments reported here the anaemic rabbits received 10 mCi of 32Pi 15 h before sacrifice. Globin mRNA was further purified by poly(U)Sepharose affinity chromatography as described by Lindberg et al. [8],except that RNA bound to the poly(U)-Sepharose column was recovered by elution with a low ionic strength buffer (buffer B) at 50 "C instead of formamide [9]. The eluate was made 0.35 M in NaCl and 2 volumes of ethanol were added. RNA precipitation was allowed to occur at -20 "C overnight. RNA was recovered by a 30-min centrifugation at 20000xg, dried under vacuum and dissolved in buffer C . Proteinase K (Merck, Darmstadt) was added up to 0.2 mg/ml and the solution incubated for 30 min at room temperature. RNA was then extracted 3 times with an equal volume of phenol/chloroform mixture. After the last extraction the aqueous phase was made 0.35 M in NaCl, RNA was precipitated with ethanol and dissolved in water at an approximate concentration of 1 mg/ml.

Globin Messcnger RNA Ageing

A1

Nylon

net

A2

C

€4

Polyacrylamide Gel Elution-Electrophoresis Polyacrylamide gel elution-electrophoresis was performed using a home-made device. The apparatus is made of plexiglass. It consists of a tube (0.8 x 30 cm) fixed by two O-rings to the part A (see cross-section in Fig. 1). Before making the gel the part A is assembled by screwing A, on Al, taking care to fit correctly the nylon net (40 mesh) against the bottom of the electrophoretic tube. This nylon net is pressed in position by two O-rings. The stopper B is then screwed on the assembled part A. The appropriate volume of acrylamide/bisacrylamide solution is then poured into the tube. After polymerization, stopper B is removed and part C is screwed in its place. As can be seen in Fig. 1, the small outgrowth on top of stopper B does not exist on top of part C. This outgrowth determines the volume of the elution chamber which is here 100 pl. It should be noted also that the nylon net is trapped in the gel after polymerization and prevents the gel from slipping u p or down, which would modify the volume of the elution chamber during electrophoresis. The perfectly well-defined volume of the elution chamber permits a perfect reproducibility. As the elution chamber has but 100 pl volume, very low elution flow rates may be used in order to ensure minimal dilution of the sample. Moreover, the apparatus described here is easy to build and use. Among the numerous polyacrylamide gel elutionelectrophoresis devices already described in the literature, none seems to offer all the above-mentioned advantages [lo-121. Fig.2 shows the device set for electrophoresis. As can be seen, the lower part of the gel is continuously washed with the buffer of the lower

-

i i i

0

1

crn

2

Fig. 1. Gal elution-elactro~horesisdevice :cross-section

reservoir, which is pumped through the hole of the stopper C, and suitable fractions may be collected at the exit of the pump. The buffer flowing across this channel ensures the electrical contact with the lower reservoir. In the experiments described in this paper 25 cm long 3 acrylamide gels prepared in 0.036 M Tris-HCl (pH 7.8), 0.03 M Na,HPO,, 0.001 M EDTA, 0.2% sodium dodecylsulphate were used [13], 50- 100 pg of globin mRNA was applied to the gel and electrophoresis was performed at 4 "C for 24 h with a constant current of 6 m A . The buffer flow reached 2ml/h; 1-ml fractions were collected. In order to prevent the formation of bubbles in the elution chamber the buffer of the lower reservoir was extensively degassed under vacuum before electrophoresis. When indicated, fractions eluted from the gel (1 ml) were submitted to molecular filtration on 10 ml G-25 Sephadex columns equilibrated in buffer A.

51 1

P. Nokin, G . Huez. G . Marbaix, A . Burny, and H . Chantrenne

Hybridization with [3H]cDNA

Gel

Fig. 2 . Gcl rlritiori-elt~cirophore.~is device set for electrophoresis

Preparation of [3HJcDNA Complementary to Globin m R N A The reaction mixture (1 ml) was 50 mM Tris-HC1 (pH 8.3), 12 mM MgCI,, 0.8 mM dATP, 0.8 mM dCTP, 0.8 mM dGTP, 130 mM NaC1, 1 mM dithioerythritol and contained 6 mCi [3H]dTTP (48 Ci/ mmol), 50 pg actinomycin D, 20 pg globin mRNA, 5 pg oligo(dT) (10 - 18 residues) and 2 pg of purified reverse transcriptase [ 141. Incubations were performed for 10 min at 37 'C. The reactions were stopped by addition of 1 ml of bufferA, and the solution was extracted with 4 ml of the phenol/CHCl, mixture. The aqueous phase was then submitted to molecular filtration on a 1-cm2x 50-cm long Sephadex G-50 column equilibrated in buffer A. The radioactive cDNA was eluted in the void volume of the column. Fractions corresponding to that region were pooled, carrier RNA from Bacillus stearothermophilus was added (20 pg/ml) the solution was made 0.35 M in NaCl and 2 volumes of cold ethanol were added. Nucleic acids were allowed to precipitate at -20 "C overnight, recovered by a 30-min centrifugation at 20000 x g and redissolved in 1 ml of 0.5 M NaOH. RNA hydrolysis was carried out for 15 h at 37 "C; the solution was then neutralized with HC1. Around 2 x lo8 dis./min of cDNA were obtained. The extent of globin mRNA transcription by reverse transcriptase was determined by hybridization of the cDNA copy with globin mRNA. After complete hybridization of equal amounts of cDNA and globin mRNA, 60 0 4of the RNA is protected against S, nuclease digestion; if twice this amount of cDNA is used, 75 of mRNA is protected.

5 pl of RNA solutions was mixed with 50 p1 of cDNA hybridization buffer containing approximately 40000 dis./min of cDNA. Hybridization was then carried out in polyethylene tubes for 18 h at 65 "C. At the end of the incubation, two 2 0 4 aliquots were taken from each sample and added to 30 p1 of a solution made of 5 pl of S, nuclease buffer (10 x ), 2 pI glycerol, 6 p1 Bacillus stearothevmophilus native DNA solution (2 mg/ml) and 17 pl of HzO.0.5 pl of nuclease S1 solution was then added to one of the two aliquots. All samples (with and without nuclease S,) were then incubated at 45 "C for 30 min. At the end of the incubation each sample was diluted with 700 p1 of 0.4 M NaCl and 250 p1 of 30% (w/v) trichloroacetic acid was added. Acid-precipitable material was collected on Millipore filters and counted by liquid scintillation. The percentage of hybridization is the ratio of acid-precipitable counts in the S,-treated sample over acid-precipitable counts in the control sample.

Hybridization with [3H]Poly(U) [3H]Poly(U) synthesis and poly(U) . poly(A)hybridization assays were performed as previously described [15].

Cell-Free Protein Synthesis Template activities of the mRNA samples were tested as previously described, using the preincubated Krebs 11 ascites tumor cell-free system [16].

Electrophoretic Separation of Globin Chains Proteins synthesized in vitro were mixed with 10 pg of rabbit hemoglobin as carrier. Acid acetone (0.12 M HC1) was added in order to dissociate globin chains and precipitation was allowed to occur at -20 "C overnight. Proteins were recovered by centrifugation at 8000 x g for 2 min, washed twice with acetone and dissolved in 50 p1 of 0.1 M sodium phosphate containing 1 % (w/v) sodium dodecylsulphate and 1 % (v/v) mercaptoethanol. a and B rabbit globin chains were separated by sodium dodecylsulphatcpolyacrylamide gel electrophoresis as described by Wood et al. [17].

RESULTS Globin Messenger R N A Fructionation Globin messenger RNA was isolated from reticulocytes of anaemic rabbits injected with [3zP]orthophosphate 15 h before their sacrifice. It should

512

Globin Messenger RNA Ageing

0.200

0

Q

0.100

0 0

10

20

30

40

50

Fraction number

Fig. 3. Gel elution-eleclrophoresis proJile of’globin 9-S m R N A . For details, see Methods. (0)Absorbance at 260 nm; (0)32Pradioactivity; ( x ) specific radioactivity determined on each RNA fraction after desalting on Sephadex G-25

be noted here that reticulocytes result from the maturation of nucleated erythroid cells in the bone marrow and the spleen. As a 10-h period elapses between the arrest of RNA synthesis in the nucleus and the release of anucleate reticulocytes in the blood stream [18], it results that in our labelling conditions only the reticulocytes which were released during the 5 last hours before sacrifice contain labelled RNA. This period of time is very short if compared to the lifetime of reticulocytes, which is about 2 days, and as a consequence most of globin mRNA isolated from those cells is unlabelled in our experimental conditions. Newly made globin mRNA molecules have thus a higher specific radioactivity than old mRNA molecules. Globin mRNA was prepared from the 15-S ribonucleoprotein released from reticulocyte polysomes by EDTA treatment, purified by sucrose gradient centrifugation and then submitted to poly(U)-Sepharose affinity chromatography. Extensive deproteinization of globin mRNA was achieved by proteinase K treatment of the poly(U)-Sepharose-bound mRNA followed by phenol extraction. This highly purified globin mRNA was submitted to elution-electrophoresis on a 25-cm long 3 % acrylamide gel. A continuous flow of buffer at the bottom of the gel gradually eluted the material coming out of the gel. The absorbance was determined in each fraction eluted from the gel and radioactivity of aliquots was measured by Cerenkov effects (Fig. 3). Globin rnRNA is eluted from the gel as a rather broad peak and the radioactivity profile is clearly shifted towards the side of the absorbance peak, which corresponds to the slowest moving molecules. The specific radioactivity of the different RNA fractions was determined after desalting on Sephadex G-25 and ethanol precipitation in order to avoid any error in the estimation of the absorbance values. (Fig.3). The fastest migrating RNA is 8- 10 times less radio-

actively labelled than the slowest one. As the electrophoretic mobility of RNA molecules is negatively correlated with their molecular weight, this result shows that newly made and ‘old’ messenger RNA molecules differ in size. Globin messenger RNA becomes progressively shorter with ageing. Globin Messenger R N A Purity The observed heterogeneity of globin mRNA could still be due to contaminations by other RNA. This possibility seemed, however, very unlikely on account of the purification procedure we used. Nevertheless, the purity of the RNA present in the different fractions eluted from the gel was tested by molecular hybridization with globin cDNA. An aliquot of each fraction was mixed with a fixed amount of [3H]cDNA and hybridization was performed in conditions where less than lo(% of the probe is annealed. Under these conditions the amount of cDNA which is annealed is proportional to the amount of homologous RNA present in the reaction mixture (Fig.4). As can be seen in Fig.5, the amount of hybridized cDNA is directly proportional to the absorbance measured in each eluted RNA fraction. Even if our cDNA probe should contain a few copies of RNAs other than globin messages, the coincidence of the cDNA hybridization profile with the absorbance profile shows that all RNA fractions eluted from the gel have the same degree of purity. Shortening of the Polyudenylute Sequence The length of the polyadenylate sequence of globin messenger RNAs was estimated in each fraction of RNA eluted from the gel by molecular hybridization with [3H]poly(U). An aliquot of each fraction was hybridized with a large excess of [3H]poly(U). The hybridization profile we obtain is clearly shifted

513

P. Nokin, G. Huez. G. Marbdix, A. Bumy, and H. Chantrenne

1000

750

-

:.

. v)

c

c

-s V

500

5x L

9 x I

1

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2

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1

4

E

L

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RNA (ngirnl)

0

Fig. 4. Linear relutionship between 9-S globin mRNA concentration and the proportion of / 3 H ] c D N A hybridized. A fixed amount (10000 counts/min) of [3H]cDNA was incubated with increasing amounts of globin 9-S mRNA for 18 h at 65 "C. For details, see Methods 30 Fraction number

Fig.6. Profile qf hybridiralion o f ' ( 3 H]poly( U ) with the R N A Jructions elurrd from the gel. A fixed amount of [3H]poly(U) (6000 counts/min) was incubated with 1 p1 of each RNA fraction for 30 min at 37 "C in 2 x standard saline citrate. For details, sce Methods. (0)Absorbance at 260 nm; (0) 3H radioactivity

0.200

conditions [15]. Taking into account the stoichiometry of the hybrid, the proportion of polyadenylate sequence in each RNA fraction was thus computed from the amount of hybridized [3H]poly(U). This proportion decreases from about 12 7; in the longest molecules to about 4% in the shortest ones. Taking 650 nucleotides as the average length of rabbit globin mRNA [19] the poly(A) stretch would correspond to about 80 and 25 nucleotides respectively.

8

?

40

0.1 00

0 30

35

40

45

Fraction number

Fig. 5. Prqfi'le of iiyhridi:ation vs /'HH]cDNA with the m R N A /ructions eluted,fiom the gel. A fixed amount (10000 countsjmin) of [-'Il]cDNA was incubated with 0.02 pl of each RNA fraction for 18 h at 65 "C. Hybridization and S, nuclease treatment were performed as described in Methods. ( 0 )Absorbance at 260 nm; (0)percentage of VHIcDNA hybridized

towards the side of the globin mRNA peak corresponding to slowly moving molecules (Fig. 6). This shows that the proportion of poly(A) in the complete molecule gradually decreases from the longest mRNA molecule to the shortest ones. We can thus conclude that the decrease in size of globin mRNA with ageing is due to poly(A) shortening. We have previously shown that a triple-stranded hybrid, poly(A) . 2 poly(U), was formed under our experimental

Trunslational Efficiency ojGlohin mRNA of Different Ages

Polyacrylamide gel elution-electrophoresis enablcd us to fractionate globin mRNA into several populations of molecules of different ages. We then measured the ability of these RNA fractions to direct c1 and fl globin chain synthesis in a Krebs I1 ascites cell-free system. The different RNA fractions eluted from the gel were desalted by molecular filtration on a Sephadex G-25 column followed by ethanol precipitation. Increasing amounts of each mRNA sample were added to a preincubated ascites cell-free extract and the incorporation of labelled leucine into proteins was measured. As can be seen in Fig. 7 the incorporation of labelled leucine into proteins increases linearly with the mRNA concentration from 0-6 pg/ml for any fraction tested. The template activity ofthe mRNA fraction shown at Fig. 8 is expressed as the radioactivity (countsjmin) of [3H]leucine incorporated in proteins

Globin Messenger RNA Ageing

514

0

6

4

2

0

Fig. 7. Stirnulotion qj’ protein .synthesis in the ascites cell-free .system by increasing concentrations of the RNA .fractions eluted from the Rel. For the fraction numbers: see Fig.8. Each point represents the mean value of two determinations. (0)Fraction 24; (W) fraction 25; (A) fraction 26; ( 0 )fraction 27; (0)fraction 28: (A)fraction 29

0.300

,

, 3

I

20

25

20

40

60

80

100

Fraction number

RNA (PgIml)

30

Fraction number

Fig. 8. Template activities of the globin mRNA fractions eluted.from the gel. Template activities are expressed in counts/min of r H ] leucinc incorporated in protein when 250 ng of mRNA is added to the cell-free system. (0)Absorbance at 260 nm; columns represent template activities; (0) ratios a/P chains of globin chains synthesized in vifro

when 250 ng of mRNA is added to the cell-free system. The longest mRNA molecules, which have been shown to be the youngest ones, are translated 2.53 times more efficiently than the shortest mRNA molecules. As SI globin mRNA is translated with a lower efficiency than fl globin mRNA in an ascites cell-free system [20,21] the difference we observe could be due to c1 and p mRNA separation during

Fig. 9. Separation of a and B globin chams by dodecylsulpliatepolj~acrylamidegel electrophoresis. For details, see Methods

electrophoresis. In order to test this possibility the ratio of a to p globin chains in the protein synthesized was determined. Protein synthesized in vitro was mixed with carrier unlabelled rabbit haemoglobin and submitted to polyacrylamide gel electrophoresis according to the method of Wood and Shaeffer [17]. Fig.9 shows an example of globin chain separation. It can be seen at Fig.8 that the c1 chainlp chain ratio does not markedly change from one side to the other of the globin mRNA peak. Consequently the observed heterogeneity in the template efficiency cannot be due to a separation of cc and fi globin mRNA during electrophoresis. It should be noted here that when unfractionated mRNA is tested in the same proteinsynthesizing cell-free system in vitro, its template activity is intermediate between that of the light and heavy mRNA molecules. Moreover, the ratio of c1 to fl chains synthesized in vitro with unfractionated globin mRNA (a/p= 0.38) is similar to the one obtained with the different mRNA fractions eluted from the gel. Therefore, the electrophoresisper se does not seem alter a or globin mRNA. Comparison oJ’ Two Globin mRNA Populations EIuted,from a Poly( U)-Sepharose Column a t Different Temperatures In order to check the preceding results, globin mRNA was fractionated into two populations differing by the length of their poly(A) stretch using a fractionation method other than gel elution-electrophoresis. Labelled globin 9-S RNA, purified by sucrose gradient centrifugation from the 15-S mRNA. protein, was submitted to poly(U)-Sepharose affinity chromatography. Unbound material which accounts for

515

P. Nokin. G. Huez. G. Marbaix. A. Burny. and H. Chantrenne

Fig. 10. Sedimrntulion profi'les ojglohin m R N A eluted at 25 'c' ( A ) and 50 ' C IS) /ion7 the poly(U)-Sc.pharose column. Radioactivity was measured on 200-p1 aliquots by Cerenkov cffect. ( 0 )Absorbance at 260 nm; (0)32P radioactivity

40

Table 1. Cliurucrcteristics of globin mRNA fractions eluted at 25 'C and 50 . CJiom u p l y ( UJ-Sepharosecolumn Template activities are expressed as counts/min of [3H]leucine incorporated in protein when 250 ng of mRNA are added to the cell-free system. Results in parentheses are arbitrary units chosen to give a template activity of 100 for unfractionated messenger R N A Poly(U)Sepharose-bound globin mRNA

Proportion

Specific radioactivity

Template activity

counts min

counts/min

s(

chains, chains

B 6 20 a

.-u

c

0

1

K5-l

Unfractionated 25 C eluted 50 C eluted

100 25 75

n.d 11.5 58

25050 (100) 17100 ( 68) 26850 (107)

0.38 0.40 0.29 "

I

0

25-30?;;', of the 9-S RNA was discarded. Globin mRNA bound to poly(U)-Sepharose was recovered by a two-step elution procedure with low ionic strength buffer at 25 "C and 50 'C. This technique separates globin mRNA molecules containing short poly(A) stretches from those containing longer ones [22]. About 25% of total bound material was eluted at 2532. Both fractions were submitted to sucrose sucrose, 18 h, gradient centrifugation (10 - 20 36000 rev./min 4 "C, SW 41) and their specific radioactivity determined (Fig. 10 and Table 1). The fractions corresponding to the hatched areas were pooled. Short poly(A)-containing molecules are 5 times less labelled than longer poly(A)-containing molecules. As in our labelling conditions the specific radioactivity decreases with the ageing of the mRNA population, this result confirms that the poly(A) stretch of globin mRNA becomes shorter with ageing.

2

4 RNA [Fgirnl)

6

Fig. 11. Slimulafion of protein .y.ynrhesis in the uscitrs cell-free system by globin m R N A jructions eluted from u poly( U)-Sep/iurose column ar 25 "C and 50 "C. Each point represents the mean value of two determinations. ( 0 ) Unfrdctionated globin mRNA; (0) 25 "C eluted globin mRNA; (A) 50 "C eluted globin mRNA

The abilities of the mRNA fractions eluted from the poly(U)-Sepharose column at 25°C and 5 0 T to direct protein synthesis in vitro were compared in the ascites cell-free system. The template activity of total globin mRNA reconstituted by mixing 25 'C and 50 "C eluted RNAs in the right proportions, was also determined (Fig. 11). The mRNA eluted at 50 ' C is 1.5 times more active than the mRNA eluted at 25 "C. The template activity of total globin mRNA corresponds exactly to the value which can be calculated taking into account the proportion and the translation efficiency of the mRNAs eluted at 25 "C and 50 "C.

516

The ratios of a to p chains synthesized in vitro in the presence of total 25 "C eluted and 50 "C eluted RNAs were determined as described above. The values of these ratios are approximately the same for the different fractions and are in the same range as those measured for the different mRNA fractions eluted from the polyacrylamide gel (Table 1). The slight difference observed between a/P ratios of the globin synthesized by the 25 "C eluted and the 50 "C eluted mRNAs cannot account for the 35 "/, difference in the incorporation of [3H]leucine into protein obtained when 25 "C eluted mRNA is used in the cell-free system instead of mRNA eluted at 50 "C. Consequently this shows that old globin mRNA molecules containing rather short poly(A) segments are translated with a lower efficiency in vitro than younger globin mRNA inolecules containing longer poly(A) stretches and confirms our previous results.

DISCUSSION The elaborate procedure of globin mRNA purification that we have used ensures a high level of purity. It has been shown indeed that the purification of globin mRNA from the 15-S m R N A . protein on one hand 123,241 and the use of affinity chromatography on the other hand [24,25] eliminates nonmessenger RNAs sedimenting in the 9-S region of the gradient. However, this highly purified globin mRNA submitted to elution-electrophoresis on a 3 % acrylamide gel is eluted as a rather broad peak. As all RNA fractions eluted from the gel hybridize with globin cDNA at the same rate, we may conclude that the purity of globin mRNA is the same in each fraction. On the other hand, with the conditions used for the electrophoresis there is no separation of the LX and p globin mRNA. Indeed, both slow and fast-moving mRNA promote the synthesis of a and p globin in the same ratio in vitro and consequently the broad peak of mRNA, which is eluted from the gel, is homogeneous with regard to a and p globin mRNA sequences. In our labelling conditions the youngest RNA molecules have the highest specific radioactivity. Specific radioactivity strongly decreases in the elutionelectrophoresis profile of globin mRNA from the slow-moving RNA species to the fast-moving ones. Consequently it appears that globin mRNA becomes shorter with ageing. [3H]poly(U) hybridization with RNA from the different eluted fractions shows that the slow-moving mRNA molecules contain polyadenylate sequences of about SO residues, whilst the fast-moving ones contain a 25-residues-long poly(A). Therefore the size difference we observe between young and old messenger RNA is due to poly(A) shortening. The shortening of the poly(A) segment at the 3' end

Globin Messenger RNA Ageing

of enkaryotic messenger RNA has been previously shown by pulse and chase labelling techniques of total messenger RNAs from HeLa [2] and mouse sarcoma ascites cells [3] and more recently for purified mouse globin mRNA [26,4]. Our results show, in addition, that both CI and fi globin mRNAs are heterogeneous in size on account of the varying length of their poly(A) tracts and have poly(A) segments in the same range of lengths. Therefore, elution of globin mRNA bound to poly(U)-Sepharose or oligo(dT)-cellulose columns by gradually increasing the temperature (Table l), the formamide concentration [27] or decreasing the ionic strength of the eluting buffer [28] do not permit a and fl rabbit globin mRNA separation. The molecular weights of a and fl rabbit globin mRNA have been estimated to be 202000 and 227000 respectively from their electrophoretic mobilities in polyacrylamide gels in the presence of formamide [29]. Such a difference, which corresponds to about 80 nucleotides, is thus not sufficient to permit a and fl mRNA separation during elution-electrophoresis on 3 sodium dodecylsulphate-polyacrylamide gels in aqueous medium. In contrast, globin mRNAs containing poly(A) tracts of either 25 or 80 nucleotides do not migrate at the same rate in the gel. It seems, therefore, that the length of the poly(A) sequence strongly influences the electrophoretic mobility of an mRNA molecule in non-denaturing conditions. The slow-down effect of the poly(A) stretch is greater than expected from the length of the sequence involved. This is in agreement with previous observations showing that poly(A) molecules 6, 28 and 84 residues long respectively migrate much more slowly than prcdicted on the basis of their size relative to 4-S and 5-S RNA [30]. For instance, the 28-residues-long molecules comigrate with the 4-S RNA in 12% sodium dodecylsulphate-polyacrylamide gels. Thus it is not surprising that the poly(A) sequence covalently bound at the 3' end of the globin mRNAs lowers the migration rate of the whole mRNA molecule much more than expected from its length. Using two different methods of mRNA fractionation we showed that young globin mRNAs have a higher template activity in a cell-free system than older mRNAs. Two phenomena seem thus to occur during mRNA ageing: a shortening of the poly(A) stretch present at the 3' end of the RNA molecule and a decrease in the ability of the mRNA to direct protein synthesis in vitro. Observations along these lines have been reported recently by Gielen et al. Using a decreasing ionic strength gradient, these authors isolated rabbit globin mRNA containing poly(A) sequence of different length from reticulocytes polysomal RNA adsorbed to an oligo(dT)-cellulosc column. Though globin mRNA fractions were still of other RNA, their contaminated by 55 - 70 results suggest that rabbit globin mRNA containing

517

P. Nokin. G. Huez, G. Marbaix, A. Burny, and H. Chantrenne

a short poly(A) stretch is translated 3-4 times less efficiently in a cell-free system than globin mRNA containing a long polyadenylate sequence. It has been clearly shown that the globin mRNA from which the polyadenylate sequence has been removed enzymically was translated in vitro as efficiently as native globin mRNA during short incubation periods similar to those used in the present work [31-331. We have confirmed that in our Krebs I1 ascites tumor cell-free extract both intact and poly(A)free globin mRNA were translated with the same efficiency in our experimental conditions (data not shown). The decreasing ability of a message to direct protein synthesis when ageing thus occurs at the same time as the poly(A) tract becomes shorter, but these two events are unrelated. The globin mRNA preparations used in the present work have been isolated from reticulocyte polyribosomes and all the message molecules may thus be supposed to be able to be translated in vivo. Among several possibilities, the molecular event(s) which make(s) the message less efficiently translatable could affect the efficiency of the initiation process. Recently published data have shown that the presence of a 5‘-terminal 7-methylguanosine is required for the translation of eukaryotic mRNAs [34,35]. However, the removal of this 5’-nucleoside or even the demethylation of the 7-metbylguanosine could not completely abolish but strongly lowered the affinity of the initiation site on the mRNA for the 40-Ssubunit Met-tRNAfM“complex. Such deficient messages, which would not be translated any more due to unefficient competition with complete message in actively metabolizing nucleated cells, could possibly be translated in old reticulocytes where newly made messenger RNA molecules have not entered the cytoplasm for several hours. It is not excluded that the loss of the 5’-terminal 7-methylguanosine occurs when mRNA ages. Work is in progress to test this hypothesis. 9

This work has been supported by a Grant from the Ministlre r k 10 Polirique ei de la Programmation Scientifiyues. P.N. holds a

fellowship of the hrstifut pour I’Encourugernent de la Recherche Scientfique tluns l’lnrlustrie et /’Agriculture. 0. H. and G. M. are fellows of the Fonds National de la Recherche Srieniifiyue.

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P. Nokin, G. Huez, G. Marbaix, and H. Chantrenne, Departement de Biologie Moleculaire, Faculte des Sciences de l’U.L.B., Ruc des Chevaux 67, 9-1640 Rhode-St-Gentse, Belgium A. Burny, Chaire de Zootechnie, FacultC des Sciences Agronomiques de I’Etat, 8-5800 Gcmbloux, Belgium