Conservation of globin messenger RNA in rabbit reticulocyte monoribosomes after sodium fluoride treatment

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CONSERVATION OF GLOBIN MESSENGER RNA IN RABBIT RETICULOCYTE MONORIBOSOMES AFTER SODIUM FLUORIDE TREATMENT. M. Toada,

J. Bavlkh , 1. W. Dvw, A. Eunh and P. A. Mahh

S. [email protected],

Department of Human Genetics Development, and Medicine Columbia University, College Physicians & Surgeons New York, N.Y. 10032

Received

April

and of

12, 1972

SUMMARY: Following NaF treatment of rabbit reticulocytes, polyribosomes dissociate to monoribosomes with concomitant cessation of protein synthesis. mRNA was isolated from different fractions of the cell before and after NaF treatment and quantitated by assay for globin synthesis in a cell-free system prepared from Krebs ascites tumor cells. mRNA activity present in polyribosomes of control cells was recovered quantitatively from monoribosomes following NaF treatment. On removal of NaF, polyribosomes reform with mRNA activity similar to that of control cells. During polyribosome dissociation, no significant amount of mRNA is recovered from ribosomal subunits, the supernatant fraction or is lost by degradation. Reticulocytes synthesis

at the

synthesize vious

ribosomes are

level

from

sodium

with

(NaF),

Thus,

mRNA for

globin

Further,

these

directly

polyribosome cating

dissociation

during

in Hela

that

with

polyribosomes

On release

is

or number cells

recovered

from NaF inhibition, 766

Copyright@1972,by

Academic Press,Inc.

treatment

(3)

cells

do not Pre-

of reticulocytes

to dissociate

into

These

removal

of NaF,

the rate

begin

to reform(l).

rate

effects

80s

(1,2).

inhibition the

these

of protein

are hemoglobins.

and polyribosomes

showed

on the size

after

cells;

that

of protein of protein

of polyribosomes and ascites

that the effect of NaF is not specific for This paper demonstrates that after treatment

associated region.

synthesis

mechanisms

formed

polyribosomes

conserved

studies

dependent

demonstrated

causes

to normal

is

studying

in mammalian

2 to 3 minutes

returns

for

90% of the protein

of protein

Within

NaF. not

than

cessation

synthesis

system

of translation

our laboratory

fluoride

reversible.

protein

a good model

RNA and more

reports

with

are

tumor

of NaF of

synthesis synthesis

by is

(2).

NaF causes

cells

(4),

indi-

reticulocytes. with NaF, the mRNA initially

quantitatively the mRNA again

in the monoribosomes becomes associated

Vol. 47, No. 4, 1972

with

BIOCHEMICAL

polyribosomes.

significant

Following

amount

or associated

NaF induced

of mRNA is

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

found

ribosomal

Kettering

II

Ascites

Laboratories,

tRNA are described from NaCl,

cells

or is

N.Y.

rabbits

kindly

The preparation paper

treated

(5).

with

of modified

when required, twice

with

washing

lysed

incubated medium,

and then

hr at 4°C to obtain in solution ribosomal 27,000

rpm,

4'C, for

gradients

were rotor

fractions SDS-method:

mRNA from were

1 mM EDTA, 0.1 M NaCl, one volume

of packed

fractions. added

the

An equal

and the mixture

total

cells

or to 5 volumes

phase

re-extracted

The resulting

aqueous

and the phenol at 4'C.

aqueous

phase

and re-extracted from

the

again final

with with

aqueous

K [0.6%

buffer, dient

pH 7.4,

was used to separate

in a SW 40 Spinco was pooled

rotor

at 4'C

volume

by adjusting

and precipitated

RNAs by spinning (5,6).

767

SDS, added

to

supernatant phase was

with

the

K for

15

first

of phenol.

The RNA

to 0.3 M NaCl,

and

by centrifugation 5 mM Tris-HCl

at 40,000

The 7s to 16s region

RNA was collected

Phenol-

was then

of Buffer

was combined

the addition of 2 volumes of ethanol. The RNA was recovered at 27,000 g for 10 min. A 5 to 20% sucrose density gradient hrs

were

The aqueous

an equal phase

(2)

supernatant

phenol

5 volumes

ribosome

(6);

ribosomal-free

30 min phase

Two methods

pH 5.01

distilled

times

sucrose

the pelleted

buffer,

at

rpm in a 65

reported

of the

of water-saturated

the

mRNA.

of the Buffer

acetate

at 4'C for

removed

at 60,000

and the ribosome-free

0.01 M potassium

rotor

from

to obtain

5

M MgClz]; for

fractions

mRNA from

with

for

in SW 27 Spinco

previously

washed

dissolved

centrifugation

10 volumes

shaken

min

was precipitated

were

SDS-method:

were

rotor

0.0015

processed

2

(5).

60 Spinco

ribosomal

M

10 mM NaF,

incubated

pellets

gradient

cells

were

described

by centrifugation

as follows:

volume

cells

and 0.13

with

cells

M KCl,

by the SDS-method

prepared

the

pH 7.4,

12 hr at 4OC and then

with

containing

incubation,

0.01

Burness,

obtained

The ribosome

Different

(1)

cell

system

cells

(2),

rpm in a Ti

density

used:

A.T.H.

were

of packed

by centrifugation

separately

mRNA were was prepared

fractions

sucrose

pelleted for

of preparing

buffer,

each experiment.

of the

and washed

as previously

at 55,000

obtained

cell-free

3 mM NaF when

ribosomes.

were using

After

prepared

M Tris-HCl

patterns

indicated Spinco

total

A[O.Ol

of Krebs

buffer

containing

an S-30

by Dr.

One volume

at 37'C.

The S-30 was centrifuged

supplied Reticulocytes

Krebs-Ringer-Bicarbonate

were

fraction

by degradation.

phenylhydrazine

7.4 mM MgC12 and 5 mM KC1 (6).

volumes

lost

no

and METHODS were

in a previous

New Zealand

NaF,

tumor

of polyribosomes,

in the supernatant

subunits MATERIALS

Krebs

dissociation

by centrifugation

rpm for

14

of the graand

Vol. 47, No. 4, 1972

washed lowed

twice

BIOCHEMICAL

with

70% ethanol

by two more washing

of Hp 0 and lyophilized. of water

for

purified

assay

rotor

for

The preparation for

protein

previously

methods

(5).

identical

reticulocyte

in a Krebs

cell

The incubation

1).

wash free

was performed

0.5 ml volume

gradient

in a SW 40

10s RNA extracted activity

fraction

system

in

10s RNA was further

stimulatory

ribosomal

ascites

density

(Figure

fol-

in a minimum

When indicated,

rpm at 4'C

had the

pH 5.5,

The RNA was dissolved

activity.

at 40,000

acetate

was redissolved

in a 15 to 30% sucrose

of rabbit

described

95% ethanol.

biological

26 hrs

synthesis

2% potassium

The RNA obtained

for

by SDS or SDS-phenol

containing

with

by centrifugation

Spinco

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were at 37'C

per

and the performed for

O.D.260. assay as

20 minutes,

1.4 1.2 10 -

-

0.6

Fig.

1:

Sucrose density gradient profile of RNA extracted from monoribosomes after NaF treatment. RNA was extracted from monoribosomes after NaF treatment and analyzed on sucrose gradients as described in "Methods" (A) Pattern obtained after 5-20% sucrose density gradient centrifugation. The shaded area including the 10s peak was assayed for biological activity. (B) Pattern obtained after 15-30% sucrose density centrifugation of the RNA present in the shaded area of (A).

768

BIOCHEMICAL

fol. 47, No. 4, 1972

and [3H]leucine

incorporation

The small tions

makes it

by direct

curve

20 min) standard

over

a range

assuming tion

of 0 to 10 pmole

an average

molecular

least

two points

The amount ponding

of

to the

were

10s RNA added weight

of RNA content into

every

set

portion

daltons

of the

(7).

for

assayed;

curve

was taken

a known amount

system

For each preparawere

standard

in a RNA preparation

of incorporation

system.

of experiments

to the 50 ~1 cell-free

of the RNA fractions

on the linear

employing protein

to the cell-free

for

of 2~10~

concentrations

10s RNA present level

10s RNA added

frac-

was determined

(incorporation

2) was determined

(5).

ribosomal

determination

activity

of purified

of 10s RNA, three

cpms were measured

mRNA content

biological

(Figure

RESEARCH COMMUNICATIONS

in different

an accurate

measurement.

relating

curve

acid-insoluble

of 10s RNA present

to obtain

density

to O.D.260

This

into

amount

difficult

optical

a standard per

total

AND BIOPHYSICAL

at

(Figure

as that

of purified

2).

corres10s RNA

on the curve.

1

‘I

0 l!L 0

L-~.I-.L-L 2

I 6

4

mRNA

Fig.

2:

I 8

10

(pmoles)

Messenger RNA concentration curve. Purified rabbit 10s RNA prepared as described in "Methods" was added to the Krebs lysate cell-free system at different concentrations and assayed as described in "Methods' [3H]-leucine (39C/mM) was used as the labelled precursor.

RESULTS: Conditions

of incubation

of reticulocytes

cause complete dissociation of polyribosomes The 40s subunits are significantly decreased The cells

were

In these

cells,

10 min

I

(4C)

then

washed

polyribosome

and was almost

with

inhibitor,

reformation

reached

after 769

employed

which

to 80s ribosomes (Figure 3,4B). after NaF treatment (Figure 3).

to remove the complete

NaF were

60 min

and incubated 20% of control (Figure

4D).

at 37'C. levels

within

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6.0 .

Fig.

from cells

3:

Effects of NaF on ribosome pattern: (A) Ribosome pattern in cells prior to incubation with NaF. The fractions indicated by shaded areas P,M and 40s were assayed for mRNA activity and the results summarized in Table I, experiment II. (B) Ribosome pattern of cells recovered after incubation with 10 u&l NaF for 60' at 37'C. The fractions indicated by shaded areas P, M and 40s were assayed for mRNA activity and the results sumP represents polyribosome region, M marized in Table I, experiment I. and 40s represents 405 ribosome subrepresents monoribosome region, unit region.

In cells

not

all

fractions,

cell

treated

monoribosome

with region

less able

than from

that

10s RNA activity

incubated NaF,

with NaF, was isolated

over

(Table

over from

75% of the mRNA activity the polyribosomes (Table

75% of the mRNA activity 1, Figure

4B).

In control

3% of the total mRNA recovered from 40s subunits or from the supernatant was not

inhibited

the cell fraction.

in preparations

18s RNA.

770

was recovered and NaF treated

recovered 1). In in

the cells,

fractions was extracIt was demonstrated containing

up to 95%

Vol. 47, No. 4, 1972

Fig.4:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Reformation of polysomes after removal of NaF. 5 ml of packed cells were incubated with 10 mM NaF for 60 min as described in Table 1. After incubation the cells were washed with washing medium containing 3 mM NaF, lysed, ribosomes pelleted and analyzed by sucrose density gradient centrifugation for 3 hrs. (A) Ribosomal pattern obtained from 5 ml of packed cells not preincubated with NaF, following the above described procedure. (B) Ribosome pattern following NaF treatment for 60 min (C) and (D) 5 ml of packed cells were incubated as in (A), the NaF removed by 2x washing with washing medium, reincubated respectively for 10 (C) and 60 (D) min in the incubation medium (see "Methods"), then lysed and ribosome pattern obtained. P represents polyribosome region, D represents dimer region and M represents monoribosome region.

10 minutes medium,

after

removal

40% of the mRNA is

60 minutes

after

control,

recovered

is

found

NaF removal, from

of NaF and incubation in the dimer

region

of cells (Table

70% of the mRNA activity,

polyribosomes

(Table

771

2, Figure

in a NaF free 2,Figure

a value 4D).

4C).

comparable

to

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Distribution

of mRNA in NaF treated Total Cells __ 40

VOLUME [ml] [I]

NaF

rabbit

reticulocytes.

-T.R.P. -

p-

450

0-D-260

M

40

sup.

-

-

-

240

30

214

1.6

-

mRNA [pmole]

510

350

40

311

14

48

VOLUME [ml]

40

-

-

-

-

240

o-D.260

0

450

203

53

4.2

-

mRNA [pmole]

490

340

248

57

15

7.2

cells; [II] - cells not preincubated with NaF. III = NaF treated Messenger RNA was obtained with the "SDS-method" from fractions P, M and 40 as indicated in Fig.3, and from the total ribosomal pellet (T.R.P.) as described in the "Methods". The SDS-Phenolmethod was used to prepare 10s RNA from the ribosome-free supernatant (Sup.). The same method was used to obtain 10s RNA fraction from total cells. The Q.D.260 represents the amount present in the ribosome fractions prior to mRNA isolation. pmoles of mRNA are obtained as described in "Methods". TABLE 2 Distribution

of mRNA in NaF treated

reticulocytes

after

Messenger P 4.8

NaF removal.

RNA [pmoles] -M -D 8.4 31.0

[II

Fluoride

[II]

10 min after

NaF removal

6.2

21.0

25.0

[III]

60 min after

NaF removal

35.0

8.2

8.4

Messenger RNA was obtained (D) and monoribosome region Fig.4. Biological activity DISCUSSION: NaF induced

inhibition

from the polyribosome region (P), dimer region (M) of sucrose gradients B, C and D shown in was assayed as described in "Methods".

of protein

ted with polyribosome dissociation AlOS RNA fraction has been shown of NaF-treated evidence

that

cells

(8).

synthesis

The present

studies

the mRNA in NaF-treated

cells

association

with

80s monoribosomes

under

synthesis.

There

is no detectable

loss

supernatant

or by degradation.

The demonstration

that

in reticulocytes

is

and accumulation of 80s ribosomes to be present in the total ribosomal is

provide

the

almost

totally

conditions

of cessation

of biologically

biologically

active

772

first

active

associa(1). pellet

direct conserved

in

of protein mRNA into

mRNA can be conserved

in

the

Vol. 47, No. 4, 1972

reticulocytes "masked

BIOCHEMICAL

without

mRNA" which

being

that

actions

eggs,

may be analogous

suggested

maternal

to exist

(12,13).

or rend,ered result

is possible inactive

in effects

potentially

similar

to those

active

Although

the rate

of protein

able

to that

in untreated

than

40% of the mRNA has become associated

rate

of protein

mRNA associated rapid

rate

inhibits elongation reports together, are

the present

compatible

a mechanism

(3,15).

with which

ACKNOWLEDGEMENTS:

cells

data

whose

expression

of

be further

This

investigation

limited

is

only

while

using

GM-18153

and N.S.F.

grant

Award

the American

supported

by N.I.H.

is

the

of a more of

NaF specifically not

affecting

study

chain

confirms

previous

of NaF treatment. cell-free

Taken

systems

of protein

(16-18)

synthesis

by

at present.

GB-27388x.

Training

than

that

reformation

(2).

was supported

Research

there

partial

initiation

defined

indicate

by the amount

the present

obtained

and less

data

shown that

chains

GM-14452,

less

is not

to inhibit

cannot

of NaF (2),

These

has been

a Faculty

an EMBO visiting

may be (14)

compar-

polyribosomes

in 40s subunits

and those

Hematology

without

to a level

with

In addition,

from

cells

factors

from NaF inhibition

a decrease

NaF acting

also

periods

these

(2).

of new polypeptide

demonstrating

mRNA in

has returned

of mRNA when there

initiation

variable

10 min of removal

It

recovery

or release (3)

within

polyribosomes.

following

the

synthesis

in these

of translation

polyribosomes

cells

egg is

of NaF in preventing

are in polyribosomes

with

animal

mRNA.

cells

synthesis

for

that

of

on erythropoiesis

by inhibitory

biologically

40% of the ribosomes

Studies

and stored

It

other

in the unfertilized

(10,ll).

temporarily

to the presence

in several

mRNA stored

fertilization

mRNA may be synthesized

translated

"masked"

translated

has been

(9) * In sea urchin expressed only after indicate

being

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dr.

Cancer

Grant

in part

by N.I.H.

Bank is Society.

TI-AM5231.

Dr.

grants

a recipient

of

Dr.

is

L.W.Dow S. Metafora

is

Fellow. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Marks, P.A., E.R. Burka, F. Conconi, W. Per1 and R.A. Rifkind. Proc. Nat. Acad. Sci. 53:1437, 1965. Conconi, F.E, A. Bank and P.A. Marks. J. Mol. Biol. 19:525, 1966. Colombo, B., C. Vesco and C. Baglioni. Proc. Nat. Acad, Sci. -61:651, 1968. Hogan, B.L. Bioch. Biophys. Acta. 182:264, 1969. Metafora, S., M. Terada, L.W. Dow, P.A. Marks and A. Bank. Proc. Nat. Acad. Sci. in press. Lockhard, R.E. and J.B. Lingrel, Bioch. Biophys. Res. Commu. 37:204, 1969. Williamson, R., M. Morrison, G. Lanyon, R. Eason and J. Paul.Biochemistry 10:3014, 1971. Gbleu, B., G. Huez, A. Burny and G. Marbaix. Bioch. Biophys. Acta -138: 186, 1967. Spirin, A.S. in Current Topics in Development Biology 1:2, 1966. Gross, P.R. and Cousineau, G.H. Biochem. and Biophys. Res. Commu.10:321, 1963.

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Vol. 47, No. 4, 1972

11. 12. 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Exptl. Cell Res. -33:368, 1964. Gross, P.R. and Cousineau, G.H. Wilt, F.H. J. Mol. Biol. 1965. -12:331, Marks, P.A. and Rifkind, R.A. -___Science 175:1955, 1972. Metafora, S., Felicetti, L. and Gambino, R. Proc. Nat. Acad. Sci.68:660, 1971 21:51, 1966Lin, S., Moesteller, D. and Hardesty, B. J. Mol. Biol. Lebleu, B., G. Marbaix, J. Chantrenne, A. Burny and G. Hzz, Bioch. Biophys. Res. Commu. 40:731, 1970. Hoers, W. andK.S. McCarty, Bioch. Biophys. Acta 228:526, 1971. Irving, J. and Hardesty, B. Fed. Proc. 31:368a,1972.

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