Selective resistance of bacterial polyadenylate-containing RNA to hydrolysis by guanosine 3′-5′-monophosphate-sensitive nuclease of Bacillus brevis

BIOCHEMICAL

Vol. 103, No. 2,1981 November 30, 1981

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 454-460

SELECTIVE RESISTANCE OF BACTERIAL POLYADENYLATE-CONTAINING RNA TO HYDROLYSIS BY GUANOSINE3'-5'-MONOPHOSPHATE-SENSITIVE NUCLEASE OF BACILLUS BREVIS Yanamandra Gopalakrishna and Nilima Sarkar Department of Metabolic Regulation Boston Biomedical Research Institute Boston, MA. 02114 and Department of Biological Chemistry Harvard Medical School Boston, MA. 02115 Received

October

12.1981 SUMMARY

Earlier experiments had shown that the degradation of newly synthesized RNA in permeable cells of Bacillus brevis is mediated primarily by a guanosine 3',5'-monophosphate-sensitive 3' -exonuclease [N. Sarkar and H. Paulus (1975) J. Biol. Chem. 250, 684-6901. More recently, we found that a substantial fraction of pulse-labeled RNA in B. brevis is polyadenylylated [N. Sarkar, D. Langley, and H. Paulus (1978) Biochemistry 17, 3468-34741, and it was thus of interest to examine the effect of polyadenylylation on Purified the susceptibility of RNA to degradation by the 3'-exonuclease. 3'-exonuclease from B. brevis hydrolyzed the unadenylylated fraction of pulse-labeled RNA from B. brevis much more rapidly than poly(A)-containing results were obtained with the pulse-labeled unadenylylated RNA. Similar Control experiand polyadenylylated RNA fractions from BaciZZus subtiZis. ments showed that the differential hydrolysis of the labeled RNA preparations by 3'-exonuclease was not due to the presence of inhibitors or activators. These results suggest that the stability of mRNA in BaciZZus species may be regulated by polyadenylylation.

The degradation

brevis is primarily

Bacillus vity

is

sensitive

(1, 2). acts

very

bacterial this

INTRODUCTION synthesized RNA in

of newly

to inhibition

The observation slowly

poly(A)-containing

B. brevis (3).

by guanosine

that

this

on poly(adenylic

mRNA might

possibility

due to the action

led

molecules High

levels

0 1981 by Academic Press, of reproduction in any form

suggested

of relatively

among the rapidly of poly(A)-RNA

0006-291X/81/220454-07$01.00/0 Copyright All righrs

(2)

Inc. reserved.

RNA from that

454

were

also

acti-

(cGMP) the

3'-end

and

the stability

of

The exploration

of

levels

(30-40%)

of

RNA species

of

high

labeled

of

whose

3',5'-monophosphate

by polyadenylylation.

to the discovery

cells

of an exonuclease

enzyme degrades acid)

be modulated

toluene-treated

found

in Escherichia

BIOCHEMICAL

Vol. 103, No. 2,198l

coZi

and BaciZZus

resemble cation, bacterial

subtilis

(4),

AND

BIOPHYSICAL

both

of which

enzyme from B. brevis

the

we compare

the

by the

harbor

3'-exonucleases

in many respects

sensitivities

RNA to hydrolysis

RESEARCH COMMUNICATIONS

(5,

of unadenylylated

6).

which

In this

communi-

and polyadenylylated from B. brevis.

3'-exonuclease

MATERIALS --AND METHODS Materials [2,8-3H]adenosine (25.5 Ci/mmole) and [5-'Hluridine (25 Ci/mmole) were obtained from New England Nuclear and oligo(dT)-cellulose (Type 7) from P-L Biochemicals. Proteinase K (Boehringer) [l'C?poly(A) and [3H]poly(U) were made from ["C]ADP (0.1 Ci/mmole and [3H]UDP (0.2 Ci/mmole) respectively as described earlier (2). The sources of all other materials are as published earlier (3, 4). Bacterial

Strains

and Growth 8185 and BaciZZus subtiZis ATCC 6051 were supplemented with salts and glucose (4).

Bacill~i~TCC nutrient

broth

grown

in

Labeling of RNA and Fractionation --Two ml of exponentially growing cultures were treated with [2,8-'Hladenosine or [5-3H]uridine (20 pCi) for 30 set and the labeled cells were processed by proteinase K method as described earlier except proteinase K was incubated for 20 min at 37" C (5mg/ml in 10 mM Tris-HCl, pH 7.5) just prior to use (4). Pulse-labeled RNA was fractionated into polyadenylylated and unadenylylated fractions by affinity chromatography on oligo(dT)-cellulose. Unadenylylated RNA was isolated from the unadsorbed fraction by precipitating 5 times with 2.5 volumes of ethanol in the presence of 10 mM MgC12. Poly(A)RNA was isolated from oligo(dT)-cellulose bound fraction by repeatedethanol precipitation in the presence of 10 mM MgClz without carrier. Labeled rRNA and tRNA were isolated from exponentially growing cultures of BaciZZus brevis that had been incubated with [3H]uridine for at least 3 generations as described (4). Nuclease from Bacilhs brevis The cGMP-sensitive nuclease was purified from BacizZus brevis cells as described earlier (2). Ribonuclease-assay ,was done by incubating labeled substrate in 0.1 M Tris-HCl, pH 7.1, 10 mM MgClz, 2 mM MnClz, .lO mM 2-mercaptoethanol and enzyme and measuring the amount of alcohol-soluble product as detailed earlier (2). The assay was done using the conditions where degree of hydrolysis was a linear function of time. A unit of ribonuclease activity was defined as the amount of enzyme that produced 1 nmole of alcoholsoluble product per min from poly(LJ). RESULTS AND DISCUSSION The relative

considerably

and tRNA were bility

of hydrolysis

from B. brevis

3'-exonuc:Lease hydrolyzed

rates

hydrolyzed

to exonucleolytic

are

of various compared

more rapidly

than

at intermediate degradation

in Fig.

1.

polyadenylic rates.

is a function

455

polynucleotides

by purified

Polyuridylic acid,

However,

acid

whereas

since

of the number

was

rRNA

the susceptiof nucleic

Vol. 103, No. 2,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

A

I

1

80.

01

ENZYME

(UNITS)

ENZYME(UNITS)

Figure

1:

Rate of exonucleolytic degradation of Hydrolysis was measured using standard cribed in "Materials and Methods," at Substrate input per assay was 0.45 mM 0.2 mM for [ 'C]poly(A) (A*.* A), 0.17 0.17 n&l for [3H]rRNA (D-3).

labeled polyribonucleotides. assay conditions as desthe enzyme levels indicated. for [3H]poly(LJ) (D-D), mM for [3H]tRNA (o-o) and

Figure

2:

(A) Rate of exonucleolytib degradation of [3H]adenosine-labeled unadenylylated RNA and [aH]adenosine-labeled poly(A)-RNA, isolated from BacilZus brevis cells. Hydrolysis was measured using standard assay condition as described in "Materials and Methods" at the enzyme levels indicated. Substrate input per assay was 1500 cpm for [3H]p~1y(A)-~A (o---o) and 1400 cpm for ['Hlunadenylylated RNA (o-0). (B) Rate of exonucleolytic degradation of [3H]adenosine-labeled unadenylylated RNA and ['Hladenosine-labeled poly(A) RNA isolated from BaciZtus sxbtilis cells. Details are as in (A). Substrate input per assay was 1250 cpm for [3H]p~ly(A)-RNA (o---o) and 1150 for [3H]unadenylylated RNA (0-o).

acid

chains,

RNA species

the relative

One can thus

substrate

of the

Exponentially

pulse-labeled

earlier

growing

fraction in Fig.

more rapidly responding

the

that not (2)

than

with

acid

is

suggested

by these

a relatively

poor

with

synthetic

but also

in comparison

with

natural

of B. brevis into

fraction

Similar

from B. subtilis (data

not

456

were

pulse-labeled

an unadenylylated

chromatography

2A, the unadenylylated

[3H]uracil

than

molecularweight

in comparison

by affinity

ENA fractions

greater

high

only

cultures

poly(A)-RNA.

with

nuclease

polyadenylic

and the ENA was fractionated

adenylylated As shown

conclude

3'-exonuclease,

as shown

adenosine

of

such as rRNA may be considerably

results.

nucleotides

activity

results (Fig. shown).

polyIWAs.

with and a poly-

on oligo(dT)-cellulose was hydrolyzed were

obtained

2B),

or with

The extent

[?I]-

(3). considerably

with

the cor-

ENA fractions of hydrolysis

of

BIOCHEMICAL

Vol. 103, No. 2,198l

polyadenylylated which

RNA did

unadenylylated

that

the

limited

about

poly(A)-RNA

other

with

fraction.

reconstitution

similar

two types

affected

fraction.

In another mixtures

unadenylylated

(Table

sensitivities are not

of

RNA were

exonuclease

type

II).

could

to exonuclease due to the presence

involved

control

I,

to

prepaappropriof

poly(A)-RNA

was sig-

of the complemen-

additivity

was observed

[3H]p~1y(A)-RNA by limiting

and [?I]amounts

show that

of poly(A)-RNA

of inhibitors

out

the hydrolysis

strict

experiments

digestion

carried

so as to permit

amounts

to hydrolysis

to be interby the presence

were

3H-labeled

acid,

3'-exonuclease

the simultaneous

in Table of

pure

in the poly(A)-RNA

experiments

of experiment,

subjected These

be produced

of equivalent

[?I]polyuridylic

that

have

RNA fractions

RNA nor

possible

described

of an activator

As shown

by the addition

thus

type

One of these

unadenylylated

is

when treated

by the

results

and unlabeled

we had observed

to degradation

of control

experiments.

?+labeled

when various

of the

It

suggests

due to contaminating

to be adsorbed

(3).

at

behavior

Indeed,

failed

or the absence

of pulse-labeled

nificantly

of 3'-

the differential

and unadenylylated

or activators

RNA

in the variousnucleic

fractions. Since

yotic

since

inhibitor

fraction.

time

experiments

This

may have been

refractory

such possibilities.

neither

acid

hand,

Accordingly,

ration

tary

a second

caution,

of a nuclease

eliminate

hydrolyzed.

fraction

may be essentially

On the

ate

15% of this

RESEARCH COMMUNICATIONS

10% even at enzyme concentrations

observed

oligo(dT)-cellulose

preted,

exceed

RNA in the poly(A)-RNA

that

with

BIOPHYSICAL

RNA was completely hydrolysis

unadenylylated earlier

not

AND

the discovery

mRNA (7-g),

stabilizing

mRNA.

the microinjection had been modified the results stabilized

of polyadenylylate

much attention Experiments into

frog

by in

vitro

have been somewhat by polyadenylylation

sequences

has been devoted to test oocytes

this

at the 3'-ends to their

possibility

of mRNA fractions

treatment

with

equivocal, (lo-12)whilethe

457

have mostly whose

appropriate

some mRNAs (e.g. stability

potential

poly(A)

enzymes. globin of others

of eucarrole

in

involved content However, mRNA)being (e.g.

of

Unlabeled

c

Alcohol-soluble

were labeled

radioactivity.

RNA fractions corresponding

prepared counterpart,

using

same methods

as

the

b Unlabeled

for

labeled

RNA and

[3H]adenosine-labeled standard condition.

+

f

using cpm.

0.3

+

+

20

46

14

44

amount

used

was equivalent

B. brevis,described in Input for 13HJunadenyl-

0.6

0.6

380 1030

1.2 0.3

+

+

172

1110

0.6

+

+

+

0.3

+

+

+

1.2

(cpm)

Substrate HydrolyzedC

(Units)

3'-Exonuclease

+

a ['HlUnadenylylated RNA and [3H]poly(A)-RNA werepreparedfrom "Materials and Methods" and nuclease digestion was done ylated RNA was 2200 cpm and for [3H]p~ly(~)-RNA was 630

to

on the Exonuclease

422

3'

246

Polyadenylylated

RNA by

RNA Fraction

0.3

Unadenylylated

Unlabeled RNA Fractionb

['HIAdenosine-labeled

Addition

0.6

of

+

Polyadenylylated

of

I

+

Unadenylylated

Radioactive RNA Fractiona

Effect Hydrolysis

TABLE

BlOCHEMlCAL

Vol. 103, No. 2,1981

AND

TABLE 3'-Exonuclea~e

Digestion

[3H]Poly(U)

of

BIOPHYSICAL

II

[3H]P~ly(U)

in

["HlUnadenylylated

RNA or

['HlPoly(A)-RNA

[3H]Unadenylylated RNA

(cd

RESEARCH COMMUNICATIONS

the

presence

of

[%]poly(~)-RNA

(cpm)

Substrate

hydrolyzed

(cpm)

(cpmf

10,000

1,220 1,246

10,000

140

1,246

1,324

10,000

1,070

10,000 Standard and either described

interferon,

dtro

entities,

experiments

with

intracellular

poly(A)-RNA therefore

degradation

cells

in the

that

bacterial

by the bacterial

polyadenyl.ylation

no longer

in bacterial

enzyme as

represented

that

thus

results

the

implicating

the

in the sensitivities

exonuclease. cells

459

cGMF'-sensitive Secondly,

of total

almost

pulse-

by the 3'-exonuclease

complete

One of the major

may thus

stability.

of rapidly

of RNA to the extent with

in

we know from

in mRNA degradation.

RXA to hydrolysis

it

subject-

bacterial

degradation

difference

endows

with

Firstly,

factor

cell

were

The

physiological

as a major

polyadenylylation

14).

of mRNA degradation

of the

Z),

(13,

the RNA fractions

is much simpler.

and unadenylylated

clear

that

of B. brevis

by cGMP (1,

all-or-none

units of substrates

of the enzymology

communication

3 '-exonuclease

labeled

naturally

2,200

the interpretation

inhibited

a nearly

is

ignorance

permeable

we find

It

and thus

to our

in this

RNA is

1,500

mRNAs) was unaffected

due to the fact

In contrast,

described

labeled

is

modification

in part

eucaryotes. mRNA

and crzu-globin

uncertainty

ed to in

L,O18

nuclease assay was done using 0.24 single or combination of labeled in "Materials and Methods."

mengovirus,

resulting

1,500

be the modulation

that

occurs

resistance functions

to of RNA

of mRNA

Vol. 103, No. 2,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENT We are grateful to Dr. reading of the manuscript.

H. Paulus for stimulating This work was supported

discussions by NIH grant

and critical GM 26517.

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Paulus, Plenum

H. and Sarkar, N. (1974) Control Publishing Corporation, New York.

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Sarkar,

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Gopalakrishna, 3545-3554.

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Lee, S. Y., Mendecki, USA 68, 1331-1335.

Langley, Y.,

H. (1975)

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G., Huez, G., Marbaix, and Cleuter, Y. (1978)

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Marbaix, G., Huex, G., Burny, A., Cleuter, Y., Hubert, E., Leclercq, M., Chantrenne, H., Soreq, H., Nucel, U. and Littauer, U. Z. (1975) Proc. Natl. Acad. Sci. USA 72, 3065-3067.

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Sehgal, P. B., Soreq, H. and Tamm, I. (1978) Proc. Natl. 75, 5030-5033. Despande, A. K., Chatterjee, B. and Roy, A. K. (1979) J. 89374942.

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Soreq, H., Littauer, E. and Chantrenne,

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Gallwitz, D., Weinberg, Nature 271, 572-573.

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