Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate

Vol. 81, No. 4, 1978 April 28,1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1145-1152

CROSSLINKING OF PROTEINS TO RIBOSOMAL RNA IN HeLa CELL POLYSOMES BY SODIUM PERIODATE Adelaide Department

Received

J.

Svoboda

and Edwin

H. McConkey

of Molecular, Cellular, and Developmental University of Colorado Boulder, Colorado 80309

March

Biology

9,1978

SUMMARY: HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was truly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18s RNA, protein L3 to 28s RNA, and proteins L7' and L23' to 5.8s RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein Sl to 16s RNA in Escherichia coli 30s ribosomal subunits, it is likely that we have crosslinked proteins to the 3'OH ends of HeLa polysomal RNAs. The study begun

(l-3).

of the topography

of eukaryotic

One can specifically

of an RNA molecule

using

identify

the technique

ribosomes

proteins of sodium

ribose

moiety.

Aldehydes

groups

of nearby

lysine

residues

to form covalent

sodium

borohydride

(4).

Proteins

Sl and IF 3, both

mRNA to the 30s ribosomal

of 16s RNA in E. coli describe

here

HeLa polysomes

small

the proteins after

sodium

subunit, ribosomal

found

there

near

can react

subunits

with

linkages

oxidation

using

important

to 18S,

this

the

3'OH end of the

the E amino

after

reduction

by

in binding

nat-

to the

3'OH end

technique

28S, and 5.8s

and sodium

recently

oxidation

have been crosslinked

crosslinked

periodate

located periodate

terminal

ural

formed

has only

(5,6).

We

RNA from

borohydride

reduct-

ion. MATERIALS --AND METHODS Cell culture and polysome p reparation: HeLa cells were grown in suspension -~culture in Eagle's minimal essential medium supplemented with 10% calf serum. Cells were labeled with [35 S] methionine (2 uCi/ml) for 24 hours in fresh medium containing 7.5 ug methionine per ml (50% of the normal methionine Abbreviations used: E. coli = Escherichia &, SDS = sodium dodecyl sulfate, CsTCA = cesium trichloroacetate, TCA = trichloroacetic acid, BSA = bovine serum albumen. 0006-291X/78/0814-1145$01.00/0

1145

Copyright 0 I978 by Academic Press, Inc. All rights of reproduction in any formreserved.

Vol. 81, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

concentration). The rate of cell growth was unaffected by this labeling cedure. Polysomes were prepared as described by McConkey (7) from cells on frozen saline and washed with buffered saline. Polysomes were stored pellets at -7OOC.

prochilled as

E. coli B were disrupted in an omnimixer as described (8). Ribosomes were harvested according to Van Duin --et al. (6). Ribosomal subunits were prepared by previously described methods (9). Crosslinking: The crosslinking procedure is a modification of previously described techniques (5,6). Unless otherwise noted, all procedures were performed at 4'C. Polysome pellets from 500 ml of log phase HeLa cells were resuspended at a concentration of 70 A260 units/ml in buffer A (4OmM NaCl, 1mM Mg acetate, 1OmM Hepes, O.lmM EDTA), pH 6.3. The solution was clarified by centrifugation at 13,000 g for 10 minutes. Polysomes were dialyzed against buffer A, pH 6.3, for 1 hour to reduce sucrose and KC1 which can react with periodate. The polysomes were oxidized by adding an equal volume of buffer A containing 40mM sodium periodate, pH 6.3, to half of the suspended polysomes. Buffer A alone was added to the remaining polysomes as a control. The samples were left in the dark for 45 minutes at room temperature. They were then chilled to 4'C and dialyzed against buffer A, pH 8.0, containing 2% glycerol, for 1 hour to inactivate the remaining periodate. The samples were then dialyzed 1 hour against buffer A, pH 8.0, without glycerol. The reduction step was carried out by the addition of l/10 volume of O.lM sodium borohydride in buffer A, pH 8.0, to both control and oxidized samples. After 10 minutes, each sample was brought, to 2% SDS and 0.5M LiCl by the addition of dry reagents. After heating at 37'C for 10 minutes, the samples were layered onto 35 ml 5-30% linear sucrose gradients containing buffer B (0.2% SDS. 3M urea, 5OmM Na acetate, 1OmM EDTA, 0.5M LiCl, and 1OmM TrisHCl, pH 5.8). Gradients were centrifuged at 67,000 g for 16 hours at 17'C. Separation of 5.8s from 28s RNA: Ethanol precipitated fractions containing the ----5.8S:28S RNA complex from the SDS-containing sucrose gradients of both control and oxidized samples were dissolved in a solution containing 0.5% SDS, 25mM Na acetate, and 5mM EDTA, pH 5.1,and heated to 60°C for 5 minutes to remove intact 5.8s RNA as described (10). The samples were then layered onto 12 ml 5-30% linear sucrose gradients in buffer B and centrifuged at 80,000 g for 18 hours at 17'C. CsTCA gradients and RNase treatment: CsTCA was prepared by titrating cesium carbonate (Alfa ?&ducts) with TCA as described for rubidium TCA (11). A combination of procedures for CsTCA gradients was used (12,13). A 5 step gradient, ranging from 2.16M to 4.34M CsTCA, was prepared in buffer C (2.5mM EDTA, 3OmM Tris-HCl, pH 8.0). Ethanol precipitated fractions from the SDS-containing sucrose gradients were dissolved in the top step of the CsTCA gradient which contained O.lM NaCl in addition to buffer C. The 5.85 RNA was loaded into the bottom step of the gradient to insure the disruption of all secondagy structure. The gradients were centrifuged at 115,000 g for 48 hours at 20 C. RNA-containing fractions from the CsTCA gradients were washed once with 0.2M NaCl in 80% ethanol. Each sample was then dissolved in 3M urea, 1OmM EDTA, 1OmM Tris-HCl, pH 7.5, at an RNA concentration of L60 A260 units/ml. One ug RNase A (EC 3.1.4.22) and 20 units RNase TL (EC 3.1.4.8) were added per A260 unit RNA. Samples were incubated at 37OC for 1.5 hours. Electrophoresis ing to Laemmli

and autoradiography: (14) using a lo-18%

One-dimensional SDS gels were run accordacrylamide gradient. Two-dimensional gel

1146

Vol. 81, No. 4, 1978

BIOCHEMICAL

analysis was a modification Fluorography was performed (17) using Kodak XR-5 film.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(15) of the Kaltschmidt and Wittmann technique (16). on radioactive gels according to Bonner and Laskey --RESULTS AND DISCUSSION

The effectiveness ribosomal

subunits.

sional

SDS gel

ribosomal

protein tion

Protein

to protein

we did that

not

there

ribosomal

seen on a one-dimen-

verifies

crosslinked

earlier

protein

experiment.

30s work

(5).

S21 to 16s RNA, a

We repeated

the observa-

to 23s RNA in periodate-treated

degradation,

(data

one protein

in Fig.

not

(to

1 which

which

procedure.

RNA) to each RNA size

50s

could

shown).

also

additional

separate

that

we reproducibly

against

3'OH ends

gradients

revealed

The fact

argues

HeLa 18s and 5;SS:

identical

18s and 28s RNA, see Fig. class

create

Furthermore,

RNA complex

represent

run after

and intact

crosslinked

3) or two proteins internal

5.8s

breaks

(to

5.8s

leading

to

crosslinks. Proteins

that

containing

sucrose

in highly

denaturing

at about

3.OM salt

from control

aggregates

removed

CsTCA gradients and RNA at about remains

bound

at the

(12).

free

are

recovered

proteins

RNA in the

2 shows

top of the gradient

protein that

whereas

of RNA (13).

SDS gels)

centrifugation

gradients,

Figure

formed

SDS-

all

protein

protein

truly

that

protein

We found during

bands

periodate

treat-

on CsTCA gradients. from

shown on one-dimensional for

In these

at the density

protein

to ribosomal

from RNA by isopycnic

3.9M salt.

(as seen on one-dimensional

The proteins

obtained

tenaciously were

to RNA sediments

ment band near

gradients

remained gradients

samples

crosslinked

are

observation

crosslinked

of the 5.85:28S

and 28s RNA peaks

additional

This

in our

of the peaks

the crosslinking

either

E. &

to 16s RNA in periodate-treated

workers

crosslinked

against

treatment

these

band

using

(5).

The sharpness

heat

shown).

was tested

prominent

crosslinked

no proteins

subunits

during

not Sl,

find

are

28s RNA argues

found

(data

procedure

Sl was the only

of protein

subunits

In addition

of our crosslinking

found

the

RNA-containing SDS gels

associated

1147

with

fractions

in Fig. RNA after

of CsTCA

3. Identical phenol

gel extraction

patterns of

Vol. 8 1, No. 4, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

m

0

1

5

IO Fraction

15

botto

02

Number

Fraction

Number

Figure 1. Sedimentation profile of 18s and 5.8S:28S RNA in 5-30% sucrose gradients containing SDS (see Materials and Methods). Periodate-treated (oxidized and reduced) and control (reduced only) profiles are identical. Figure 2. Protein distribution in CsTCA density gradients. Ethanol precipitates of sucrose gradient fractions containing 18s RNA (Fig. 1) were diss lved in CsTCA and centrifuged as described in Materials and Methods. Cpm [ 33 S] met in control (o- - -0) and periodate-treated (o o) 18s RNA fractions.

crosslinked

particles

proteins tein

in Fig.

spots

removal

3 is

29,000 this

protein

linked

in Fig.

dalton

during

protein

both

is

protein

other

also

it

It

appears

gels,

the pro-

due to the is

studies

clear

incomplete

from our

of mammalian

low methionine The 18,000

to resolve of its

location

to be L23'

1148

gel

data,

ribosomes polysomes.

to 28s RNA. The approximately

RNA appears

assignment.

because

of the

two-dimensional

in two-dimensional

apparently

difficult

analysis

to 18s RNA in periodate-treated

to 5.8s

the L7'

gel

presumably

electrophoretic

crosslinked

and its

However, data),

tails,

to resolve

substantiate

electrophoresis.

4. In these

L3 can be crosslinked

difficult

RNA is

A two-dimensional

the RNase treatment.

crosslinked

gels

to 5.8s

(unpublished

with

protein

shown).

displaced

S3a is

show that

one-dimensional data)

shown

of nucleotides

that

We also

not

show anodically

and from a comparison (18),

(data

(19).

clearly

to

be L7' gels, content

dalton

(19). its

Although

migration

in

(unpublished protein

cross-

by two-dimensional

and low methionine

content

gel

BIOCHEMICAL

Vol. 81, No. 4, 1978

a

b

AND BIOPHYSICAL

c

d

e

RESEARCH COMMUNICATIONS

f

BSA

RNase

Figure 3. SDS acrylamide gel of proteins crosslinked to ribosomal ENA after CsTCA gradient sedimentation. The RNA has been removed by ENase treatment as described in Materials and Methods. BSA was added to each sample as carrier for the precipitation of protein in cold 20% TCA before the addition of SDS sample buffer. The gel was stained with Coomassie blue. a) HeLa polysomal proteins b) control 18s ENA c) periodate-treated 18s ENA d) control 5.8s: 28s ENA e) periodate-treated 28s ENA f) periodate-treated 5.8s RNA.

A rough we crosslink

calculation protein

ure correlates

well

to about with

IF3 to 16s EEA in E. coli whether

sodium

reducing

agent

borohydride in our

indicates

that

lo-152 found

(6).

in all

that,

of the available for

RNA species ENA molecules.

the periodate-induced

Essentially

or sodium

three

the

same results

cyanoborohydride

experiments.

1149

(20,21)

studied, This

fig-

crosslinking were

obtained

was used as a

of

Vol. 81, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 4. Two-dimensional gel analysis of proteins found crosslinked to RNA. Non-radioactive 80s ribosomal proteins were always added to radioactive samples to insure the correct fluorogram spot identification. a) Coomassie blue b) periodate-treated 18s RNA stained pattern of HeLa polysomal proteins c) periodate-tre ted 28s RNA d) periodate-treated 5.85 RNA- Frame b is a fluorogram of [ 39 S] met labeled proteins from RNA in CsTCA gradients. Frames c and d are fluorograms of proteins recovered from RNA in the SDS-containing sucrose gradient separating 5.8s from 28s RNA. Patterns identical to, but fainter than, frames c and d have been obtained for periodate-treated 5.85 and 28s RNA after CsTCA gradient treatment.

Although

it

is most

3'OH ends of RNA molecules, internal

RNA bases

to form aldehydes tide

bases.

However,

likely

that

we have

the

possibility

must be considered. (22)

which recent

could data

crosslinked

that

we have

Glycoproteins crosslink

suggest

1150

there

crosslinked

can react

to RNA via that

proteins

amino

is only

with groups

to the true protein periodate on nucleo-

one glycoprotein,

to

Vol. 81, No. 4, 1978

BIOCHEMICAL

a 31,000

on the

ulocyte form

dalton

species (23).

An N-terminal

an aldehyde,

which

might

(24).

These

excluded

by our

data.

Protein position

on Kaltschmidt is

then

return

view

of the marked

atectomy in this

helpful

in rats event

for

is

elevation

quite

interesting.

Our thanks

of Health

internal

to his research

preprint

as a rat

liver

two hours

protein role

on a protein

ribosomal

of partial

after

synthesis

the donation

on RbTCA gradients,

was supported

by a grant

can also

cannot

be

to the

same

protein

hepatectomy,

hepatectomy following

of S3a and of the

for

retic-

in RNA, upon periodate

RNA crosslinking

by 18 hours

to L. Gold

group

and rabbit

to 18s RNA, migrates

gels

of liver

the possible

This

an amino

within

position

(26),

access

advice.

Institutes

and Wittmann

liver

or threonine

can be crosslinked

original

Acknowledgements: W. Bauer

for

seen to move cathodically to its

serine with

possibilities

S3a, which

which

react

RESEARCH COMMUNICATIONS

in chicken

60s subunit,

ribosomes

treatment

AND BIOPHYSICAL

(25). partial

In hep-

3'OH end of 18s RNA

of 3.

coli

cells,

and to C. Cantor from

and

to for

the National

(GM 21749). REFERENCES

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BIOCHEMKAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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