The translational capacity of deadenylated ovalbumin messenger RNA

Cell, Vol . 8, 5 1 -58, May 1976, Copyright ©1976 by MIT

The Translational Capacity of Deadenylated Ovalbumin Messenger RNA M . T . Doel and N . H . Carey G . D . Searle & Co . Ltd . Research Laboratories Lane End Road High Wycombe Buckinghamshire, HP12 4HL, England

We present evidence that the poly(A) sequence at the 3' end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system . Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact . It is shown that the stability of the mRNA in a cellfree system is not appreciably affected by this procedure. Measurements of the size of ovalbumin-synthesizing polysomes, rate of peptide elongation, and number of rounds of translation per messenger show a generally reduced efficiency for deadenylated mRNA compared to native mRNA . No comparable difference was observed in experiments with a wheat germ cell-free system, which gives few rounds of translation per mRNA . This indicates that the effect results from a lowering of the efficiency of reinitiation on deadenylated mRNA .

1973) . There are also the possibilities that the poly(A) has some effect on the translational capacity of the mRNA or is necessary for the binding of specific proteins or other factors (Singer and Penman, 1973 ; Blobel, 1973) . Experiments with a total mRNA population from L cells (Bard et al ., 1974) and with mouse globin mRNA (Williamson, Crossley, and Humphries, 1974) have indicated that deadenylated mRNA is capable of being translated with an efficiency close to that of native mRNA in systems from wheat germ and Krebs ascites cells . However, the work of Huez et al . (1974) and of Marbaix et al . (1975) indicated that the poly(A) is a factor in the functional stability of mRNA, and Gielen, Aviv, and Leder (1974) have provided evidence that mRNA with a long poly(A) is translated more efficiently than mRNA with a shorter stretch of poly(A) . This report describes an examination of the effect of removing the poly(A) sequence from ovalbumin mRNA on its ability to be translated in two cell-free systems, the rabbit reticulocyte lysate and the wheat germ S30 system . In the wheat germ system, the rate and extent of ovalbumin synthesis from native and deadenylated mRNAs were very similar . In the reticulocyte lysate, however, differences were detected in the rates of elongation and the average number of rounds of translation on each messenger . The deadenylated mRNA was markedly less efficient in this system than the native mRNA .

Introduction

Results

The presence of poly(riboadenylic acid) segments at the 3' terminus of messenger RNA (Edmonds, Vaughan, and Nakazato, 1971 ; Darnell, Wall, and Tushinski, 1971 ; Lee, Mendecki, and Brawerman, 1971), heterogeneous nuclear RNA (Nakazato, Kopp, and Edmonds, 1973 ; Greenberg and Perry, 1972), and mitochondrial RNA (Perlman, Abelson, and Penman, 1973 ; Ojala and Attardi, 1974) is well established . This poly(A) segment is added posttranscriptionally (Perry, Kelley, and LaTorre, 1974), and it is reasonable to suppose that it fulfills some specific function in the expression of the mRNA . Philipson et al . (1971) suggested that the poly(A) functions in transport of mRNA from nucleus to cytoplasm, and Sussman (1970) and Sheiness and Darnell (1973) postulated a role in determining the mRNA lifetime . Histone mRNAs lack a poly(A) segment (Greenberg and Perry, 1972 ; Adesnik and Darnell, 1972), as do some nonhistone mRNAs in sea urchin embryos (Nemer, Graham, and Dubroff, 1974) . These messengers are quite functional, and studies on mRNA turnover suggest that there is little or no difference between histone mRNA and adenylated mRNA (Greenberg, 1972 ; Singer and Penman,

The use of 3'-OH-specific exonucleases to remove the poly(A) from mRNA has been described by Williamson et al . (1974) for purified mouse globin mRNA and by Bard et al . (1974) for an mRNA population from L cells . We have used polynucleotide phosphorylase (E .C .2 .7 .7 .8 .) to deadenylate purified ovalbumin mRNA . This RNA has been purified from laying hen oviduct to apparent homogeneity on acrylamide gel electrophoresis . In some of the earlier experiments, we used mRNA of the type shown in Figure 1(l), which contained some 18S rRNA . In the case of mRNA of the type depicted in Figure 1(11), over 85% of the labeled peptides synthesized in a wheat germ cell-free system were precipitated by a monospecific anti-ovalbumin antibody (M . E . Haines, unpublished results) . In studies of this sort, it is important to use polynucleotide phosphorylase free of any other nuclease activity . All preparations examined were nuclease-free as assayed by methods relying on the production of acid-soluble material . However, we examined the enzyme by a more sensitive assay involving incubation with rat liver polysomes . After incubation, the polysomes were displayed on a su-

Summary



Cell 52

crose gradient, and the degree of nuclease contamination was reflected in degradation of the larger polysomes . Figure 2 shows two such assays . Any batches of enzyme which showed profiles such as that in Figure 2(11) were rejected . Control experiments showed that 0 .001 units of ribonuclease T1 was detectable by this assay . Our criterion for the loss of the poly(A) sequence was 2 fold : loss of the ability to bind to oligo(dT)cellulose and loss of the ability to hybridize in solution to 3 H-poly(U) (data not shown) . Neither of these rules out the possibility that up to 5 or 6 adenylate residues remain at the 3' end of the molecule . Table 1 shows the amount of mRNA activity binding to oligo(dT)-cellulose after a series of digests with polynucleotide phosphorylase . The bound activity decreased rapidly over the first 20 min and had almost disappeared after 40 min . The rate of phosphorolysis of homopolymers [especially poly(A)] by polynucleotide phosphorylase is very rapid compared to the rate of phosphorolysis of heteropolymers (Grunberg-Manago, 1963) . The enzyme should therefore digest the mRNA less rapidly once it reaches the heteropolymer sequence adjacent to the poly(A), but it is quite possible that some of the untranslated sequence presumed to lie between the 3' end of the coding sequence and the poly(A) may also have been removed . We attempted to examine this possibility by hybridizing native mRNA and deadenylated mRNA to 3 H-labeled cDNA prepared using AMV reverse transcriptase (gift of Dr . Beard, Duke University) under conditions of RNA excess . If a portion of the mRNA beyond the poly(A) has been removed, part of the 5' end of the cDNA will be unable to

1

gas

1 s

I

28c

1ss

5

5

l

S vyYl 5

hybridize, and the final extent of hybridization will be lower than for native mRNA . Table 2 shows the results of such an experiment, and it is plain that there is no significant difference between native and deadenylated mRNA . The cDNA is estimated to be 1000-1100 nucleotides long (data not shown), and so a difference of 1 % in the degree of hybridization would correspond to a loss of 10 or 11 bases ; we therefore conclude that we have lost less than this number of bases from the untranslated region of the mRNA . The deadenylated mRNA was examined by electrophoresis on acrylamide :agarose gels . The messenger activity profiles of RNA recovered from such gels are shown in Figure 3, and two points are worthy of note . The activity in material which binds to oligo(dT)-cellulose always resides in material of the same molecular weight as intact mRNA, and the

10 . MIGRATION (cm)

.

~"~'

5

16

Figure 1 . Electrophoresis of Purified Ovalbumin mRNA Preparations on 2 .4% Acrylamide :0 .4% Agarose Gels (I) mRNA after one cycle of binding to oligo(dT)-cellulose and sedimentation through sucrose gradient showing OD profile (solid line) and mRNA activity profile (dashed line) . (II) mRNA recovered from gel shown in (I) and reelectrophoresed under the same conditions . Arrows show the positions of rRNA markers run on parallel gels .

SEDIMENTATION

- ,I-

Figure 2 . Use of Polysomes to Check for Nuclease in Polynucleotide Phosphorylase Preparations (I) Polysomes incubated for 30 min with no enzyme . (II) Polysomes incubated with polynucleotide phosphorylase containing traces of nuclease . (III) Polysomes incubated with nuclease free polynucleotide phosphorylase .



Translation of Deadenylated Ovalbumin mRNA 53

activity which appears in the unbound fractions has moved to a position of lower molecular weight very early in the digestion, but does not subsequently move further . Formamide gel analysis of material from a digestion of highly purified mRNA, for which the optical density and mRNA activity profiles were consistent throughout, is shown in Figure 4 . The molecular weight of the deadenylated mRNA was estimated to be about 1 .6 x 10 5 daltons lower than that of intact mRNA . If taken at its face value, this corresponds to the loss of about 500 nucleotides . It has been estimated by gel electrophoresis that the length of the poly(A) sequence in total oviduct mRNA preparations may exceed 500 residues (Dornan, Cook, and Carey, 1974), but estimates of only 44 residues (Shapiro and Schimke, 1975) or 70 residues (Rosen et al ., 1975) have been made by hybridization assays for the poly(A) sequence of purified ovalbumin mRNA . This difference has been shown to be due to the markedly anomalous migration of large adenylate oligomers on polyacrylamide gels (Woo et al ., 1975) . The measurements of the size of the ovalbumin synthesizing polysomes and of ribosome transit times were made using mRNA which contained some residual 18S rRNA, since these measurements are independent of the amount of mRNA added . The polysome sizes and the way in which

they changed with time were similar to the results of Palmiter (1973) in the case of native mRNA, as shown in Figure 5(1-III) . The sizes are in general smaller than those reported, but this is probably a function of the reticulocyte lysate used, and in any case, this does not affect comparative measurements done with the same batch of lysate . For native mRNA, the maximum ovalbumin messenger activity coincided with 7 ribosome polysomes after 10 min of incubation ; after 30 min, the size had dropped to 6, and after 70 min, to 2 or 3 . The measurements with deadenylated mRNA, shown in Figure 5(IV-VI), showed rather broader distributions of messenger activity . The maximum activity coincided with 6 ribosome polysomes after 10 min of incubation, and this dropped to 5 after 30 min, and

Native mRNA

Deadenylated mRNA (Not Bound to

(Bound to Oligo(dT)cellulose) 28s

01 igo(dT)cel lu lose)

18s

e

e

e

e

28s

18s

;

',

5

1

Table 1 . Loss of the Ability of mRNA to Bind to Oligo(dT)-Cellulose after Incubation with Polynucleotide Phosphorylase Time of Incubation

Activity Bound to Oligo(dT)-Cellulose

Total Activity Not Bound to Oligo(dT)-Cellulose

0 min

8 .20 x 106 cpm

4 .10 x 104 cpm

20 min

2 .31 x 104 cpm

4 .72 x 106 cpm

40 min

1 .30 x 104 cpm

6 .50 x 106 cpm

60 min

0 .91 x 104 cpm

5 .34 x 106 cpm

5

10

5

10

15

10

5

10

5

10

5 e

The values listed are 3H cpm incorporated into ovalbumin in a 90 min incubation at 25°C in the reticulocyte lysate . They are the product of the counts incorporated per ug of added RNA and the total number of ug in the fraction .

E . 0 U 2

Table 2 . Hybridization of 3H-Labeled cDNA to Native and Deadenylated mRNA

RNA cDNA

Native mRNA

Deadenylated mRNA

50 ng

50 ng

0 .5 ng

5

'

,

No mRNA Blank

Time of Hybridization

55 hr

55 hr

cDNA in Hybrid

69 .2%

70 .1

I,

e

5

0 .5 ng

10

10

0 .5 ng 55 hr 1 .7%

-Mean value of three determinations . The RNA used was homogeneous on gel electrophoresis . Hybridization for 55 hr gives a Crot of 3 at the concentrations used .

MIGRATION a ( cm .) Figure 3 . Electrophoresis of Polynucleotide Phosphorylase Digested mRNA on 2.4% Acrylamide :0 .4% Agarose Gels Samples were digested for 0 min (upper panels), 10 min (center panels), or 20 min (lower panels), and bound to oligo(dT)-cellulose . The profiles are of ovalbumin mRNA activity recovered from the sliced gels . Migration is from left to right .



Cell 54

to 2 after 70 min . Thus it seems that the polysomes translating deadenylated mRNA are somewhat smaller than those translating native mRNA . The measurements of ribosome transit time involve looking at incorporation of radioactivity in the reticulocyte lysate into total acid-precipitable material and into ovalbumin as judged by the immunoprecipitation assay . The horizontal displacement between the curves is equal to one half the transit time (ti/,) (Fan and Penman, 1970) . Measurements were made of the initial transit time by having label present from the start of the incubation and of the transit time after 30 min by adding the label 30 min after the start of the incubation . The results are

shown in Figure 6 . The t i/, values after 30 min are essentially the same as the initial values, and in both cases, the t, for deadenylated mRNA is about 1 min greater than for the native RNA . This indicates that the rate of elongation is lower by about 20% on the deadenylated messenger . The rate of elongation, in terms of amino acids added per second, is given by L/ It _ r f, where L is the polypeptide chain length (387 in the case of ovalbumin), t is the transit time in seconds, and r is the time necessary for the release of completed chains . It has been shown by Lodish and Jacobson (1972) that r is small (of the order of 15 sec) in the case of globin, and if this is also the case for ovalbumin in the reticulocyte lysate, it will have little effect on the calculation when transit times are 500-600 sec . The calculated figures are shown in Table 3 . From the data in Table 3, it is possible to calculate the number of times each mRNA is translated on average during a given incubation period . This was done according to method B of Palmiter (1973), which does not

20

4

Figure 4 . Formamide Gel Electrophoresis of Deadenylated mRNA (I) Native mRNA . (II) Deadenylated mRNA . Gels were scanned in the ultraviolet (solid line) and sliced . The dashed line shows the ovalbumin mRNA activity in RNA recovered from the slices . (III) Estimation of the molecular weight difference between native and deadenylated mRNA . Markers were rRNA from chicken oviduct and E . coli run on parallel formamide gels.

f o

/

IU so

10

10

5

34

40

1a

2 0

30

40

Incubation Time (min)

FRACTION

nurses

Figure 5 . Measurement of the Size of Ovalbumin-Synthesizing Polysomes in the Reticulocyte Lysate (I-III) Reticulocyte lysate programmed with native ovalbumin mRNA, incubated for (I) 10 min ; (II) 30 min ; or (III) 70 min . (IV-VI) Reticulocyte lysate programmed with deadenylated ovalbumin mRNA, incubated for (IV) 10 min ; (V) 30 min ; (VI) 70 min . Solid line is the OD260 profile . Histogram is the ovalbumin mRNA activity in each gradient fraction expressed as cpm incorporated into ovalbumin % cpm incorporated into globin

Figure 6. Determination of the Ribosome Transit Time on Native and Deadenylated mRNA Samples were withdrawn from standard incubations at the times indicated and assayed for incorporation of 3H into ovalbumin (0-0) and into total acid-precipitable material (0- •). (I) Native mRNA ; label added at t = 0 min . (III) Native mRNA; label added at t = 30 min . (II) Deadenylated mRNA ; label added at t = 0 min . (IV) Deadenylated mRNA; label added at t = 30 min .



Translation of Deadenylated Ovalbumin mRNA 55

Table 3 . Parameters of Ovalbumin Synthesis in the Rabbit Reticulocyte Lysate Native mRNA

Deadenylated mRNA

Initially

After 30 Min

Polysome size (P)

7

6

Transit time (t)

9 .8 min

9 .6 min

Elongation rate (387)

0 .66 as/sec

0 .67 as/sec

0 .56 as/sec

0 .55 as/sec

Teff (P )

0 .71

0 .52

0 .54

0 .45

\t Number of Rounds of Translation

Initially

After 30 Min

6

5

11 .6 min

In 30 min-13 In 70 min-29

11 .8 min

In 30 min- 8 In 70 min-19

These parameters are defined in the text .

depend upon knowing the amount of mRNA present . This calculation gives values of 29 for native mRNA and 19 for deadenylated mRNA after 70 min of incubation . Palmiter (1975) has defined the translational efficiency J e ff) of an mRNA as the number of complete ovalbumin peptides released from each polysomal mRNA per minute . Under steady state conditions, Teff = Pit, where P is the polysome size, and t is the ribosome transit time . Calculated values for Teff are given in Table 3, and it will be seen that the values for native and deadenylated mRNA have dropped markedly in the first 30 min of incubation . The values for deadenylated mRNA are lower at all times, that is, this mRNA is translated less efficiently than native mRNA . We have examined the synthesis of ovalbumin with time, in response to the addition of highly purified ovalbumin mRNA, in the reticulocyte lysate, and in a wheat germ S30 system . From standard incubation mixtures, aliquots were withdrawn at various times and examined for 3 H incorporation into immunoprecipitable ovalbumin in the case of the reticulocyte lysate, or 35 S incorporation into acid-precipitable material in the case of the wheat germ S30 . The results are shown in Figure 7 . As may be seen from Figure 7(l), there is a considerable difference in the extent of incorporation, expressed as cpm incorporated per ug of RNA added, between native and deadenylated mRNA . The incorporation of 3H into globin was effectively the same in both cases . This difference, of about 35%, is not seen when the two mRNA preparations are translated in the wheat germ system . After 90 min of incubation in this system, there is only a 6-7% difference in incorporation between native and deadenylated mRNA, and from this experiment alone it might be concluded that both mRNA preparations are identically efficient . To examine the possibility that deadenylated mRNA is degraded more rapidly in the reticulocyte lysate, a further time course experiment was performed . Standard mixtures were incubated with native or deadenylated mRNA for 3 hr, by which time

15-

Z

-

Ir

E

Native mRNA~

Deadenylated mRNA

n.i

•

Native mRNA

i 10-

d m

I

a 0

Deadenylated mRNA

•

E a

5.

50 INCUBATION TIME (min)

100

Figure 7 . Comparison of the Translation of Native and Deadenylated mRNA in the Reticulocyte Lysate and the Wheat Germ S30 System (I) Translation in the reticulocyte lysate . Solid lines show incorporation of 3H into ovalbumin per µg of added mRNA . Dashed lines show incorporation of 3H into globin . (II) Translation in the wheat germ S ao . Solid lines show incorporation of 35S into acid-insoluble material per ug of added mRNA . Dashed line shows incorporation of 35S in an incubation with no added mRNA .

the synthetic capacity of the reticulocyte lysate is exhausted . The synthesis of ovalbumin was followed as before . Then fresh lysate was added and

Cell 56

synthesis was allowed to proceed for a further 2 hr . As may be seen from Figure 8, ovalbumin synthesis recommenced in both incubations, and in both cases proceeded to an extent of 82% of the level observed in the first incubation period . This would indicate that the cessation of incorporation in the reticulocyte lysate is not due to a lack of active mRNA . Thus it seems that the native and deadenylated mRNAs are degraded to the same extent over a 5 hr period in a cell-free system . The difference between the translational efficiency of the two species cannot be accounted for by differential degradation . Discussion The role of poly(A) in the function of mRNAs to which it is attached remains unclear . Work on polio virus RNA has shown that loss of the poly(A) has a marked adverse effect on the infectivity of this RNA (Spector and Baltimore, 1974) . If it were to be assumed that the poly(A) in viral messenger RNA has the same function as the poly(A) in eucaryotic cytoplasmic mRNA, then the poly(A) should be necessary for the translation of at least some species of eucaryotic mRNA . However, studies with deadenylated mRNA (Williamson et al ., 1974 ; Bard et al ., 1974 ; Sippel et al ., 1974), together with the ability of mRNAs naturally lacking poly(A) (for example, histone mRNA) to be translated, have shown that the poly(A) tract is not absolutely necessary for translation . Earlier studies on the translation of deadenylated messengers were carried out using protein-synthesizing systems from wheat germ (Roberts and Paterson, 1973) or Krebs ascites cells (Mathews, 1972), which give at most a few rounds of translation per message . Thus they are insensitive to differences affecting reinitiation or slight lowerings of translational efficiency which only become apparent over longer periods of translation . A study of deadenylated mouse globin mRNA using a system from rabbit reticulocytes, which is known to translate exogenous mRNA many times during a prolonged period (Palmiter, 1973), showed that reinitiation is possible on deadenylated mRNA (Humphries, Doel, and Williamson, 1974) . It was estimated, however, that the deadenylated mRNA was only translated 11 times in a 90 min period, compared to 19 times for control mRNA-a reduction of 42% . The significance of this difference is not easy to assess, since the method used to calculate the number of rounds of translation depended upon an estimation of the number of active mRNA molecules present in the sample . Our study in the rabbit reticulocyte system of the efficiency of translation of deadenylated ovalbumin mRNA showed a similar dif-

ference between adenylated and deadenylated species . After 70 min of incubation, we calculated that there had been 10 fewer rounds of translation on the deadenylated mRNA-a reduction of 35% . The ribosome transit times measured on deadenylated mRNA are lower at all times than those for native mRNA . This suggests that the apparent rate of elongation is lower by about 20% on the deadenylated mRNA . This would lead to larger polysomes if the initiation rate remained constant, whereas the contrary is observed . Thus the results point to a decreased rate of initiation on the deadenylated mRNA . If all the mRNA remains intact during the incubation and all the ribosomes which initiate produce complete polypeptides, then the parameter Teff is equivalent to the rate of initiation . In the reticulocyte lysate, the ovalbumin mRNA has to compete throughout the incubation with endogenous globin mRNA for a limited pool of various protein factors . In such a competitive situation, it is not improbable that small changes in factorbinding efficiency could cause large changes in the rate constants of synthesis . A role for the poly(A) in binding specific factors therefore remains a strong possibility (Blobel, 1973) . It has been report-

Figure 8 . Translation of Native and Deadenylated mRNA in the Reticulocyte Lysate over an Extended Period After 3 hr, fresh lysate was added with fresh 3 H-isoleucine to keep the specific activity of the incubation mixture constant . (I) Incubation from t = 0-180 min . (II) Incubation from t = 180-360 min . These counts have the t = 180 min values subtracted from them .

Translation of Deadenylated Ovalbumin mRNA 57

ed that rabbit globin mRNA competes with ovalbumin mRNA in a Krebs ascites system to the same extent, regardless of the presence or absence of the poly(A) tract (Sippel et al ., 1974) . This, however, was a comparison of two exogenous mRNAs in a poorly reinitiating system, and the system in which such comparisons are carried out may greatly affect the conclusions which are drawn . As is shown in Figure 7(l), there is a considerable difference in the extent of incorporation between native and deadenylated mRNA in the rabbit reticulocyte system . The ratio of incorporation (native/deadenylated = 1 .61) after 70 min of incubation corresponds well with the ratio of the number of rounds of translation in 70 min (native/deadenylated = 1 .53) . The fact that the deadenylated mRNA is able to be translated in the wheat germ system at an efficiency comparable to that for native mRNA indicates that the difference observed in the rabbit reticulocyte system is due to some intrinsic property of the deadenylated molecule which causes it to be recognized differently in this system, and not merely some nonspecific degradative phenomenon . The results indicate that the poly(A) in ovalbumin mRNA, while not completely essential, has a direct influence on the efficiency of translation . Our data do not enable us to say at which point in the synthetic sequence the effect is exerted, but they do seem to indicate a lowering of the rate of initiation on the deadenylated messenger . It is interesting to note that in a recent paper, Nemer, Dubroff, and Graham (1975) showed that sea urchin embryo mRNAs lacking poly(A) are less loaded with ribosomes than adenylated mRNAs in vivo . They concluded that intracellular messengers lacking poly(A) initiated at a slower rate than adenylate messengers . Experimental Procedures Purification of Ovalbumin mRNA RNA was purified from total oviduct RNA from laying hens (Dornan et al ., 1974) or from oviduct polysomal RNA obtained by precipitation with magnesium (Palmiter, 1974) . One cycle of binding, in a batchwise procedure, to oligo(dT)-cellulose (Searle Products for Research) followed by sedimentation through 5-22 .5% isokinetic sucrose gradients (18 hr at 25,000 rpm, Beckman SW 27 .1 rotor) produced a 50 fold purification of mRNA activity . This RNA which, as shown in Figure 1(1), contains small amounts of 18S rRNA, was further purified by electrophoresis on 2 .4% acrylamide:0 .4% agarose gels . After scanning in a Joyce-Loebel ultraviolet gel scanner, the portion of the gel containing the mRNA was excised, and the RNA was recovered by homogenizing the gel in buffer as described by Haines, Carey, and Palmiter (1974) . This mRNA migrated as a single peak on reelectrophoresis, as shown in Figure 1(II) . Its mRNA activity was enhanced about 100 fold with respect to polysomal RNA . Assays of mRNA Activity Ovalbumin mRNA activity was assayed in a rabbit reticulocyte lysate as described by Palmiter (1973) . 5,6- 3 H2-isoleucine (15 Ci/m

mole, Radiochemical Centre, Amersham) was used as the labeled amino acid at 10 pCi/ml . Ovalbumin synthesis was measured by incorporation of 3H into a specific immunoprecipitate. The wheat germ S30 cell-free system was prepared by the method of Roberts and Paterson (1973) . Incorporation of 35S-methionine (>100 Ci/m mole, Radiochemical Centre, Amersham) into acidprecipitable material in response to added mRNA was monitored . Polynucleotide Phosphorylase Digestions Polynucleotide phosphorylase (E .C .2 .7 .7 .8 .) from M . luteus was the gift of Dr . M . Eaton (Searle Laboratories) . A typical reaction mixture contained, in 1 ml, mRNA 100 µg, 15 mM sodium phosphate, 50 mM Tris-CI (pH 8 .2), 15 MM MgCl 2 , polynucleotide phosphorylase 0 .5 units (1 unit is defined as liberating 1 µmole of ADP from poly(A) at 37°C) . After incubation at 37°C, the mixture was made 0 .2% in sodium dodecylsulphate and extracted with 2 vol of phenol :chloroform (1 :1) . The aqueous layer was washed twice with an equal volume of chloroform, then precipitated overnight with 2 vol of ethanol at -20°C . The precipitate was spun down (20,000 x g for 10 min) and redissolved in 500 mM KCI, 10 mM Hepes (pH 7 .5) to give an RNA concentration of 100 ,ug/ml. This material was bound to oligo(dT)-cellulose (100 mg for 100 µg of RNA) and washed 4 times with 1 ml of 500 mM KCI, 10 mM Hepes, once with 1 ml of 100 mM KCI, 10 mM Hepes, and 4 times with 1 ml of 10 mM Hepes . The combined 500 mM KCI washes (deadenylated mRNA) and the combined 10 mM Hepes washes (native mRNA) were precipitated with 2 vol of ethanol at -20°C . Hybridization to cDNA 3 H-labeled cDNA to purified ovalbumin mRNA was prepared using AMV reverse transcriptase as described by Cox, Haines, and Emtage (1974) . The specific activity of the cDNA was 7 x 106 cpm/µg . Hybridizations were performed in siliconized glass capillaries as described by Cox et al . (1974) for times long enough to ensure complete hybridization (up to a Crot of 3) . The percentage of the cDNA in hybrid was determined using S1 nuclease . Measurement of the Size of Oval bum! n-Synthesizing Polysomes This was done by displaying the polysomes on sucrose gradients and assaying across the gradient for ovalbumin mRNA activity, as described by Palmiter (1973) . Measurement of Ribosome Transit Time Transit times were obtained as described by Palmiter (1973), by comparing the incorporation of label into immunoprecipitable ovalbumin to the incorporation into total acid-precipitable material . 10 ml incubation mixtures were used, containing 100-150 pg of native or deadenylated mRNA . Comparison of mRNA Stabilities in the Reticulocyte Lysate Reticulocyte lysate incubation mixtures (2 ml) were set up containing 20 pg of native or deadenylated mRNA . Aliquots were removed at intervals up to 3 hr and assayed for ovalbumin in the usual way . After this time, fresh lysate was added to the residual mixture and incubation was continued for a further 3 hr . Aliquots were removed at intervals and assayed for ovalbumin . Assay for Traces of Nuclease in Polynucleotide Phosphorylase Rat liver polysomes were prepared by a magnesium precipitation method (Palmiter, 1974) . 5 OD2 60 units of polysomes were incubated at 37°C for 30 min with the polynucleotide phosphorylase preparation (0 .5 U/ml) in 25 mM Tris-CI (pH 7 .5), 25 mM NaCl, 5 MM MgC1 2 . Under these conditions, the enzyme does not attack mRNA . The polysomes were displayed on a linear 0 .5-1 .5 M sucrose gradient in the same buffer . The absorbance profile was compared with one from polysomes incubated in the same way without enzyme .

Cell 58

Gel Electrophoresis Polyacrylamide/agarose composite gels (0 .6 x 10 cm) were prepared as described by Loening (1967) . Formamide gel electrophoresis was performed on 4% acrylamide gels as described by Haines et al . (1974), except that a running buffer of 36 mM Tris-CI (pH 7 .7), 20 mM NaH 2 PO4 , 1 mM EDTA was used . All gels were run at a constant 5 mA/gel at 20°C . Acknowledgments We would like to acknowledge the expert assistance of G . H . Catlin and E . A . Cook during the course of this work . We also wish to thank Dr . A . J . Hale for providing excellent research facilities .

Perry, R . P ., Kelley, D . E ., and LaTorre, J . (1974) . J . Mol . Biol . 82, 315-331 . Philipson, L ., Wall, R ., Glickman, G ., and Darnell, J . E . (1971) . Proc . Nat . Acad . Sci . USA 68, 2806-2809 . Roberts, B . E ., and Paterson, B . M . (1973) . Proc . Nat . Acad . Sci . USA 70, 2330-2334 . Rosen, J . M ., Woo, S . L . C ., Holder, J . W ., Means, A . R ., and O'Malley, B . W . (1975) . Biochemistry 14, 69-78 . Shapiro, D . J ., and Schimke, R . T . (1975) . J . Biol . Chem . 250, 1759-1764 . Sheiness, D ., and Darnell, J . E . (1973) . Nature 241, 265-268 . Singer, R . H ., and Penman, S . (1973) . J . Mol . Biol . 78, 321-334 .

Received November 11, 1975 ; revised January 19, 1976

Sippel, A . E ., Starrianopoulos, J . G ., Schutz, G ., and Fiegelson, P . (1974) . Proc . Nat . Acad . Sci. USA 71, 4635-4639 .

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