Two-dimensional polyacrylamide gel electrophoresis separation of low molecular weight nuclear RNA

Vol. 56, No. 4, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS SEPARATION OF LOW MOLECULAR WEIGHT NUCLEAR RNA

Ramachandra Reddy, Department

Thomas O. S i t z ,

of Pharmacology, Houston,

Tae Suk R o - C h o i and H a r r i s Baylor

College

Busch

of Medicine

T e x a s 77025

Received January 2,1974 SUMMARY

Two-dimensional electrophoresis successively on 10%, pH 7.2, and 12%, pH 3°3, polyacrylamide gels was used to separate nuclear and whole cell 4-8S RNA fractions from Novikoff hepatoma ascites cells into individual RNA species. With this method, analytical studies were possible on two new species of RNA referred to as 4.2S RNA and 4.5S RNAII , respectively. The 4.2S RNA has the nucleo-

t i d e c o m p o s i t i o n AMP, 1 8 . 0 ; UMP, 2 3 . 9 ; GMP, 2 7 . 9 ; and CMP 2 6 . 7 . T h i s RNA c o n t a i n s 2 ~MP r e s i d u e s a n d an a l k a l i stable dinucleotide. The 4 . 5 S RNAII was d i s t i n c t l y s e p a r a t e d f r o m 4 . 5 S RNAI and 4 . 5 S RNAII I a n d h a s t h e n u c l e o t i d e c o m p o s i t i o n AMP, 2 0 . 6 % ; UMP, 2 3 . 7 % ; GMP, 3 0 . 0 % ; a n d CMP, 2 5 . 7 % . I t h a s no %MP o r 2 ' - 0 methylated nucleotides. In addition, several other spots were s e p a r a t e d f r o m t h e m a j o r RNA s p e c i e s . INTRODUCTION Single dimensional polyacrylamide gel electrophoresis has been extensively used in this and other l a b o r a t o r i e s individual RNA species

(i).

Recently,

to

fractionate

two-dimensional polyacryl-

amide gels have been used to achieve greater resolution of proteins (2), RNA and partial digests of RNA (4).

(3) and transfer RNA

Earlier studies from this laboratory have shown that nu-

clear 4.5S RNA and 5S RNA obtained from preparative polyacrylamide gel electrophoresis were separated into several components on DEAE-Sephadex columns

(5,6).

The two-dimensional polyacryl-

amide gel electrophoresis method presented here provides a more satisfactory separation of these RNA species and, several new minor RNA species were found.

Copyright © 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

1017

in addition,

Vol. 56, No. 4, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS Labeling for

of

incubation

Nuclear

of

orthophosphate,

of

RNA

by

density

sucrose

Separation

w/w)

were

of

10%

4-8S

RNA

0.04

M

Tris,

blue

marker

0.5

run,

in

RNA

The

second

40

per

were

in

was

with

an

40

tained

x 40

from

dimension

from

the the

15

acid night

first

marker

first

was

to migrates

kept

in

at

starting

room

30-35

After

the

pH

3°3

o

The

two

clean

labeled

point

first

6

M

of

dimension

urea

second

the

in

mA

from

the

plates

the

40

until

spots

strips

were

3.3

strip.

cm

the

first

strip

pH

cold

buffer The

room

gel

ob-

before

acrylamide

the

The

12~

long

acid

around

-

dimension

glass

6 M urea-citric

or

of

strips

Soaking

RNA

the

the

gel

electrophoresis.

cm.

in

The

The

40

pH

bromphenol

concentration

temperature

at

highly The

from

used

acetate,

and

am

polymerization

electrophoresis

Acrylamide

buffer

sodium

accommodated

the

(5).

autoradiography.

w/w)

solution.

the

gel,

acrylamide

between

weight

earlier

The

sucrose.

at

dimension°

dimension

the

by

run

readily

positioned

improved

before

subjected

cm,

acrylamide

min

gel

0.3

the were

pouring

x

M

4°C.

identified

molecular

(I).

0.02

5%

procedure

bisacrylamide:acrylamide,

30-35

at

bisacrylamide:acrylamide,

gels,

in

slot

gel

dimension acid

made

32p_

Two-Dimensional

slabs

EDTA,

with

described

(1:39,

to migrate

mA

bands

M citric

(1:39,

the

high

by

em

procedures

cells

been

gels

M

- The

SDS-phenol

from

have

x 0.3

Vitro

the

Fraction

were

allowed

at

the

0.025

for

placed was

application,

RNA

0.002

wide

by

fraction

x 20

cm

In

ascites

RNA

polyacrylamide 40

was

of

RNA

4-8S

as

Slots

32p

gradient

made

contained 7.2.

4-8S

by

METHODS

hepatoma

extraction

separation

- The

RNA

Novikoff

and

Gels

AND

over-

slabs

bromphenol identified

were blue

by

autoradiography° Extraction extracted

and

and

Nucleotide

precipitated

Composition from

the

1018

of

acrylamide

RNA gel

- RNA

was

as

described

Vol. 56, No. 4, 1 9 7 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

j U-3

QU'-2 h

U:-I 5S

5S ]TI

C

4.5S~

$STI 4.5SI ~.5SlI 4S

Fig. Io Autoradiograph of 4-8S nuclear RNA separated by two-dimensional polyacrylamide gel electrophoresiso The first dimension gel contained 10% polyacrylamide and was run for 40 hr in a buffer containing 0°04 M Tris-acetate, 0.002 M EDTA, and 0.02 M sodium acetate, pH 7.2; the second dimension gel contained 12% polyacrylamide and was run for 40 hr at pH 3.3 in 0.025 M citric acid containing 6 M urea.

earlier for of

(7).

16-18 %MP

hr

and

The at

in

37°C

and

2'-0-methylated

electrophoresis graphy

air-dried

the

at

pH

second

RNA the

was

hydrolyzed

nucleotide

in

the

dimension

0.25

compositions,

nucleotides 3.5

with

were

first

the

determined

dimension

N NaOH

and

content

by chromato-

(8,9).

RESULTS Separation separated

earlier

did not s e p a r a t e procedure

of 4 o 5 S RNA - A l t h o u g h on D E A E - S e p h a d e x as a d i s c r e t e

separated

like 4.5S RNA I (i0)

4 . 5 S RNAII does

the 4 . 5 S RNA's

A-50

peak.

columns

However,

as a d i s t i n c t

not c o n t a i n

1019

(5),

were the 4o5S RNAII

the t w o - d i m e n s i o n a l

spot

(Fig°

i) which

~ M P or 2 ' - 0 - m e t h y l a t e d

Vol. 56, No. 4, 1 9 7 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 2. A u t o r a d i o g r a p h o f 4 - 8 S w h o l e c e l l RNA s e p a r a t e d two-dimensional polyacrylamide gel electrophoresis. The c o n d i t i o n s w e r e t h e same a s d e s c r i b e d f o r F i g u r e 1.

nucleotides

(Table I).

The n u c l e o t i d e c o m p o s i t i o n s

and 4.58 RNAII I are very similar 4.2S RNA - In addition,

to those r e p o r t e d

(Fig.

AMP+UMP+~MP/GMP+CMP

(Table I) which

cantly greater

of 4.5S RNA I earlier

4~2S RNA was visualized

spot between 4S RNA and 4.58 RNAs ratio of 0.81

than that of whole

i).

by

(5).

as a distinct

This RNA has a

transfer RNA

is signifi-

(ii).

Separation of 5S RNA - The three 5S RNAs were separated earlier by c h r o m a t o g r a p h y

on D E A E - S e p h a d e x

5S RNAII and 5S RNAII I (6). clearly RNA

separated

(Fig.

results

The t w o - d i m e n s i o n a l

these 58 RNA species

I) or whole cell 4-8S RNA

compositions

of these three species

published

earlier

columns

into 5S RNAI,

procedure

from either nuclear 4-8S

(Fig. 2).

The n u c l e o t i d e

(Table I) compare well

to

(12).

Several minor spots labeled A, B and C (Fig. i) were found that were not observed electrophoresis.

p r e v i o u s l y by o n e - d i m e n s i o n a l

slab gel

The nucleotide a n a l y s e s of these species

1020

Vol. 56, No. 4, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

TABLE NUCLEOTIDE

COMPOSITIONS

I

RNA'SS E P A R A T E D

OF N U C L E A R

BY TWO-DIMENSIONAL

GEL E L E C T R O P H O R E S I S AMP+UMP+* MP

RNA

AMP

UMP

GMP

CMP

~ MP

NmpNp

4.2S

18.0

23°9

27.9

26.7

2.4

1.3

0.81

4o5S I

25.7

22.8

22.6

28.9

-

-

0.94

4.5SII

20.6

23.7

30.0

25.7

-

-

0°80

4.5Sii

I 23.5

24.1

17.7

16.3

3.6

14.9

1.51

5S I

18.5

23.7

30.2

27.8

-

-

0.73

5SII

18.6

23.5

30.6

27.3

-

-

0.73

5SII I

21.8

32.0

21.1

19.7

1.6

3.8

1.36

A

23.6

32.0

22.0

22.6

-

-

1.25

B

2201

29.2

24.8

23.9

-

-

1.05

C

20.3

26.2

27.7

25.8

-

-

0.87

The nucleotide compositions are percentages the RNA (8,9). The values are averages of two employing different RNA preparations.

indicate

that

nucleotides

they

do

(Table

not

contain

any

*MP

GMP+CMP

of the total 32p in different experiments

or

2'-0-methylated

I)o

DISCUSSION The

increased

acrylamide of

the

minor fer on

gel

4.5S

5S

A,

B and

region.

The of

the

basis

phoresis

at

and

base

composition

are

protonatedo

pH

obtained

electrophoresis

and

spots RNA

resolutions

RNAs, C,

chain 3.3

the and

first

appearance the

length.

because low

pH

the

apparent

The

of

the

RNA

the

adenine

and

6 M urea

also

1021

separation

4.2S

RNA in

the

RNA

dimension

the

and

on

the

observed

separates second

two-dimensional

from

separation

dimension

separates

The

is

by

the

basis

cytosine disrupt

the

trans-

mainly

of of

and

electro-

chain

length

residues base

pairing

Vol. 56, No. 4, 1 9 7 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and aggregation that might affect the secondary structures of these molecules. This method is very useful for the separation of individual RNAs from a mixture of RNAs as shown by its separation of the 4-8S RNAs or for isolating partial digestion products of an RNA for determining RNA sequence.

It is possible that it will also

be of value for separation of low molecular weight messenger RNA species and tRNA as shown by studies currently in progress in this laboratory. ACKNOWLEDGMENTS The authors wish to thank Rose K. Busch for supplying the tumor transplanted animals, Charles W. Taylor for incubating the tumor cells with 32p-orthophosphate in vitro and Dale Henning for his skillful technical assistance.

This work was supported

by USPHS grant CA-I0893.

R EFER ENC ES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12o

R o - C h o i , T. S . , C h o i , Y. C . , S a v a g e , H. E . , and B u s c h , H . , I n , Methods in C a n c e r R e s e a r c h (H. B u s c h , E d . ) , V o l . IX, New York, Academic P r e s s , pp. 71-153 ( 1 9 7 3 ) . O r r i c k , L. R . , O l s o n , M. O. J . , and B u s c h , H . , P r o c . N a t . Acad. S c i . U . S . A . 7 0 , 1316 ( 1 9 7 3 ) . D e W a c h t e r , R . , and F i e r s , W., A n a l . B i o c h e m . 4 9 , 184 ( 1 9 7 2 ) . I k e m u r a , T . , and D a h l b e r g , J . E . , J . B i o l . Chem. 248, 5024 (1973). R o - C h o i , T. S . , Moriyama, Y . , C h o i , Y. C . , and B u s c h , H . , J . B i o l . Chem.~ 245, 1970 ( 1 9 7 0 ) . M o r i y a m a , Y., I p , P . , and B u s c h , H . , B i o c h e m . B i o p h y s . A c t a 209, 161 (1970) o S h i b a t a , H . , Reddy, R . , H e n n i n g , Do, R o - C h o i , T. S . , and Busch, H., Mol. Cell. Biol. In press. Wyatt, G. R°, Biochem. J. 48, 584 (1951). Hirsch, D., Jo Mol. Biol~ 5---8,439 (1971) o Ro-Choi, T. S., Reddy, Ro, Henning, D., Takano, T., Taylor, C. W., and Busch, H., J. Biol. Chem. 247, 3205 (1972). Galibert, F., Larsen, C. J., Lelong, J. C., and Boiron, M., Nature 207, 1039 (1965). Ro-Choi, To S., Reddy, R., Henning, D., and Busch, H., Biochem. Biophys. Res. Commun. 44, 963 (1971) o

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