Vol. 170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1990

Pages

QUANTITATION

J.W.

OF ENDOGENOUS

Backus,’

M. J. J.D.

LIVER APOLIPOPROTEIN

Eagl eton,’

Sparks,’

S.G.

and H.C.

2,3

Harris,

513-518

B mRNA EDITING

C.E.

Sparks,’

Smith 1,2,3,*

Departments of ’ Biochemistry , and ’ Pathology and Laboratory Medicine and 3 the Cancer Center , University of Rochester, Rochester, NY 14642

Received

May 8, 1990

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo Bk) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo 8, mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited. ~1990 AcademicPress, Inc.

Apolipoprotein

B (apo B) is a single copy gene product

either a 512 kDa protein B48 in humans (l-3).

Editing

codon

(@A)

at position

rise to apo B, (l-3).

2153 is converted

Consequently,

mRNAs

Although regulation

the

molecular

mechanism(s)

data suggest

secretes

whereas

human

apo B, in chylomicrons

*Corresponding

lipoprotein

of C-->lJ, stop codon

transitions

mRNA editing responsible

Following (VLDL).

liver secretes

known

wherein

(apo

as RNA

a glutamine

&J!AA) thereby

giving

half of apo B, and apo B, i s identical. (4), whereas

(6) involve different for apo

that it is tissue-specific

(11,12).

very low density

(13,14),

the amino terminal

mRNAs also involves C-->lJ

(8-10) and hormonally-regulated

as VLDL

a transition

to a translation

(5) and paramyxovirus

are unknown,

form primarily

and apo B, in rats) or a 250 kDa protein

of apo B mRNA involves

of plant mitochondrial

mitochondrial

from apo B mRNA as

and apo B, in rats) due to a novel form of RNA processing

editing

Editing

(apo Bl 00 in humans

translated

synthesis,

B mRNA

kinetoplast motifs,

editing

and

its

(7) and both developmentallyapo B assembles

with lipid to

In rat liver both forms of apo B are secreted only

and VLDL (7). Alterations

apo B, VLDL of secretion

(1516).

Rat intestine

of edited and unedited

author. 0006-291x/90 513

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

forms

BIOCHEMICAL

170, No. 2, 1990

of VLDL apo B could

concentrations (minutes

as the half-lives

versus

oligonucleotide

have significant

hours,

endogenous

editing

yield similar

quantitation

consequences

RESEARCH COMMUNICATIONS

to steady

This report

chain

reaction

compares

hybridization

(competimer-PCR)

radiolabeled

probes

are used

apo

deoxy

for quantitating

that the two techniques

in conjunction

B

different

and competing

techniques

activity on rat tissue RNAs. The data suggest when

state plasma

of VLDL apo B, and VLDL apo B, are dramatically

ref. 13).

polymerase

AND BIOPHYSICAL

can

with competing

deoxy oligonucleotides. METHODS Livers from normal rats were quick frozen and pulverized in liquid nitrogen, and Dounce homogenized in 8M guanidine HCI. Rat small intestines were extensively flushed in situ (blood supply intact) with ice cold buffered isotonic saline before removal and quick freezing. Insoluble material was cleared by centrifugation and RNA differentially precipitated with a half volume of ethanol. RNA was resuspended in guanidine HCI and ethanol precipitated several times until the 260/280 absorbance ratio was 1.8 or greater. The final RNA pellet was resuspended in diethyl pyrocarbonate treated water. Apo B cDNA was prepared from 2 pg of total liver or intestinal RNA using 5 I.IM ND2 deoxy oligonucleotide (ND2, GTTClllTTAAGTCCTGTGCATC, has its 5’ end at nucleotide 6718 of both edited (STOP) and unedited (GLN) apo B mRNAs) as reverse transcriptase primer. Reverse transcriptase and reaction conditions were from Promega. Apo B STOP and GLN cDNAs were amplified by the polymerase chain reaction (PCR) using 2.5 units of Taq polymerase (Promega) and ND2 and ND1 deoxy oligonucleotide primers (ND1 , ATCTGACTGGGAGAGACAAGTAG, has its 5’ end at nucleotide 6512 of both STOP and GLN apo B mRNAs). The cycle conditions were: one cycle of 30 set at 94°C 2 min at 55°C and 1 min at 94”C, 2 min at 55°C and 1 min at 70°C. At the end of these cycles the reactions were held for 5 min at 30°C to ensure complete conversion to double stranded 206 bp PCR product. The PCR buffer was 10 mM Tris (pH=8.5), 2.5 mM MgCI,, 50 mM KCI, 0.1 mg gelatin/ml, 0.05% NP-40, 0.05% Tween-20 and 800 PM dNTPs. All PCRs were carried out with a HybaidTM thermal cycler. Quantitation of STOP and GLN PCR products by differential hybridization was essentially as described by Davidson et al. (11). Agarose gel resolved 206 bp PCR products were transferred to nylon membranes (BioRad), prehybridized in hybridization buffer minus probe and hybridized with end-labeled deoxy oligonucleotide probes BGLN and BSTOP at 41 ‘C (BGLN, TACTGATCAAA-TATC, and BSTOP, TACTGATCAAATTTATC; both have their 5’ end at nucleotide 6679 of apo B mRNA). GLN and STOP blots were then washed at 48°C and 46°C respectively, to discriminate for specific hybridization (11). Polynucleotide kinase and reaction conditions for labeling 5 pmols of deoxy oligonucleotide ends were from Promega. All deoxy oligonucleotides were purchased as gel purified DNAs from Genetics Design Inc., Houston, TX. Quantitation of STOP and GLN PCR products by competimer differential hybridization was as described above except that probes were added with 1 OO-fold excess of the respective competing BSTOP or BGLN deoxy oligonucleotide. Lastly, competimer-PCR was used to quantitate STOP and GLN 206 bp PCR product essentially as described by Gibbs et al. (17). To selectively amplify a 167 bp PCR GLN product, the initial PCR reaction described above was diluted lOOO-fold into a second PCR containing 0.5 PM each BSTOP and BGLN and 5 pmols end-labeled BGLN (held at 94’C for 1 min while adding the target 206 bp PCR fragment) followed by 8 cycles of: 15 set at 94”C, 1 min at 45°C and 1 min at 70°C. Reactions were held for 5 min at 5O’C after the eighth Cycle to ensure completion of double stranded 167 bp products at high affefic specificity. Selective amplification of PCR STOP products was carried out as described for GLN except the end-labeled primer was BSTOP. The 167 bp fragments from one tenth of each reaction were resolved on agarose gels. Blots and gels from the competimer-PCR were exposed to XAR-5 film and subsequently quantitated by direct scintillation counting of excised bands. 514

Vol.

170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS Quantit,ation

of edited and unedited

apo B mRNAs (STOP and GLN mRNAs respectively)

was carried ‘out using three different protocols in common mRNAs

three batches

using

fragment

flanking

Quantitation oligonucleotide

through

chain

BGLN and BSTOP A, lanes

l-4)

was at 41 “C for both

All procedures

thermal

washes

essentially

(PCR) using

had

of apo B of a 206 bp

ND1 and ND2 deoxy

(see Methods).

hybridization

of 5’ end-labeled

deoxy

1, see Methods

for sequences)

to the 206

as described

by Davidson

et al. (11).

but the differential

of 46’C

by amplification

6666 as primers

involved (Figure

probes

followed

reaction

the edit site at nucleotide

(panel

discriminative

,234

as primer,

by the first two approaches probes

bp PCR product Hybridization

oligonucleotide

cDNA by the polymerase

oligonucleotides

Methods).

of total rat liver RNA and their initial reverse transcription

ND2 deoxy

from

(see Experimental

and 48’C

hybridization

for BSTOP

was controlled

and BGLN respectively

5676

-206bp

Ethidium bmmide

Southern

Slots

0

1 Figure 1. Panel A: Lanes 1-4 show ethidium bromide stained 206 bp PCR products amplifiedfrom total liver RNA. A Southern transfer of this gel was cut into individual lanes and processed by differential thermal washes after hybridization as described in Methods. Lanes 5-8 of Panel A were: hybridized with BSTOP and washed 46°C hybridized with BGLN and washed at 48’C, hybridized with BSTOP with competimer BGLN and washed at 46°C and hybridized with BGLN with competimer BSTOP and washed at 48°C. Panel B: Lanes l-4 show ethidium bromide stained 167 bp PCR product resulting from the competimer-PCR with: end-labeled BGLN using PCR amplified genomic DNA as starting material, end-labeled BGLN using PCR amplified genomic DNA as starting material, end-labeled BGLN using PCR amplified genomic DNA as starting material and end-labeled BGLN using PCR amplified genomic DNA as starting material. The end-labeled BSTOP and BGLN primers which were not extended during the PCR appear at the bottom of lanes 5-8 and serve to demonstrate that equal cpms went into each reaction. The percent apo B mRNA as GLN and STOP were calculated by dividing the sum of cpms for BGLN and STOP for a given analytical condition into the cpms for BGLN and BSTOP alone X 100. Figure 2. Lanes 1 and 2 show ethidium bromide stained 167 bp BSTOP and BGLN PCR product (respectively) resulting from the competimer-PCR amplification of small intestine 206 bp PCR product as described in Figure 1, Autoradiograph of the gel is shown in lanes 4 and 5 for BSTOP and BGLN respectively. The percent apo B mRNA as STOP and GLN were calculated as describd in Figure 1 and is indicated on the respective lanes. The position of the 167 bp PCR product and end-labeled oligos are the same as in Figure 1.

515

Vol.

No. 2, 1990

170,

(11).

BIOCHEMICAL

Autoradiography

hybridized BSTOP edited

and

(sum of cpms and BGLN

direct

respectively.

hybridization

BSTOP).

BSTOP

Figure deoxy

(n=3

with excess

was identical

cold

are

suggested

due to

that the range

to the first with the exception simultaneously

and end-labeled

BGLN

in panel A, lanes 7 and 8 in Figure

used

yield a 167 bp PCR product

to compete

(lanes

l-4 of panel

apo B mRNA was 54-63%

DNA 206 PCR products

BSTOP:BGLN

with

of

that a

with the probe with

excess

1. Quantitation

B).

another

cold

of these

during

and BGLN

a second

contain

PCR

technique)

Using the competimer-PCR

rat livers). only

BSTOP

for competimer-PCR

to

technique,

B). The range

of values

As a control

in the competimer-PCR,

CAA) were

reamplified

ratio of 3:97 (lanes 4 and 5 of panel B, demonstrating

competimer-PCR

where

(lanes 7 and 8 of panel

(n=3

(which

method

one

(see Methods

60% of the total apo B mRNA was unedited

and gave

the high specificity

a

of the

technique.

Competimer-PCR

was also used to quantitate

total RNA (Figure

206 bp product

2).

Following

(lanes

BSTOP

synthesis

3 and 4) and scintillation

total apo B mRNA was edited

and BGLN

PCR amplification

was subject to competimer-PCR

2). Autoradiograph intestine

A), 55 and 45% were

was added

BGLN

of the 206 bp PCR product

for unedited

counts

rat livers).

1, panel B shows data from the third quantitative

amplification

intestinal

1, panel

that of the total

that 67% of the total apo B mRNA was unedited.

oligonucleotides

genomic

indicated

by this method

deoxy oligonucleotide

The results are shown

data suggested

counting

Quantitation

approach

1 OO-fold excess of competing (i.e. end-labeled

scintillation

in lanes 5 and 6 of Figure

apo B mRNA was 50-65%

The second

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of intestinal

cDNA, the resultant

of the 167 bp product

counting

and edited

apo B mRNA in small

showed

respectively

(lanes 1 and

that 85% and 15% of

(+/- 5%, n=3).

DISCUSSION Southern

blot hybridization

hybridization methods

with discriminative

with discriminative

for determining

liver. The respective

thermal

thermal washes

the percent

quantitation

and competimer-PCR

of unedited

of unedited

washes,

Southern

blot competimer-

have been compared

as

apo B mRNA in total RNA from normal

rat

apo B mRNA determined

by these three methods

was 45, 67 and 60% of the total rat liver apo B mRNA. Literature quantitative

values for rat liver STOP and GLN mRNA levels vary widely.

methods,

here were collected different

methods

A potential washes

STOP:GLN on common

preparations

difficulty

errors can be introduced

from an amplified

of rat liver RNA and therefore

population

(cloning

also suggest

that

quantitation.

of error in quantitation

due to the technical

Furthermore,

ratios range from 87:13 (3) to 13:12 (11). The data presented

alone can yield different source

Using very different

might be introduced

in controlling

precise

temperatures

whenever

a technique

or PCR).

An assumption

516

during

selects

the discriminative during

the washes.

a category

of molecules

explicit

in the quantitation

of

Vol.

170, Nc. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

PCR products

is that the ratio of mRNAs remains

constant.

provided

the number

low

that

of PCR cycles

amplification

of one mRNA plateaus,

up” (17,18).

The number

We suggest based

on

B mRNA,

Assuming

that the translation

obtained

and unedited

molar ratio of apo B variants

be 1 :I (apo B H:L).

to avoid

rate is uniform

mRNAs have different

loading

Competitmer-PCR secretes

primarily

RNA editing

apo B, (7,19).

alone.

play a major

results from cellular secret primarily

role.

rates and/or

which

15% unedited

is quite different

alone

and of apo

“loading,”

the

can be calculated

to

of rat liver apo

that either GLN and STOP

significant

regulation

of intestinal

in intestine

even

occurs

though

of intestinal

secreted

from the ratio of 1:6 B&B,

translational,

An additional

heterogeneity

mRNA

Direct measurements

Once again therefore,

regulatory

rat liver

at the

levels.

revealed

a mass ratio of 1:20, B,:B,

in normal

Direct measurements

a mass ratio of 1:2 for H:L (7,19), suggesting

and secretory

(see Methods).

twice the size of apo B, the predicted

to be 2:l.

and translation

to “catch-

over the entire length

on RNA availability

Given that apo B, is approximately

where

competimer-hybridization

B have demonstrated

post-translational

is allowed

mRNA do not differ in ribosome

based

mass ratio flor apo B H:L can be calculated

by

to be true

a situation

60% of the total apo B mRNA is unedited

in quantitation

and that the edited

theoretical

enough

while the other has not and therefore

consistency

competimer-PCR.

This has been shown

of PCR cycles have been limited for this reason

that on average

the

are

RESEARCH COMMUNICATIONS

possibility,

post-translational however,

tissue (intestinal

this tissue

apo B suggest suggested

and secretory

by

control

is that 15% unedited

mRNA

stem cells, for example

which

B, ref. 20).

Acknowledgments: The authors wish to thank Jenny M.L. Smith for preparation This work was supported by the Cffice of Naval Research grant N00014-89J1915 HCS) and PHS ROIHL 29837-06 (awarded to CES).

of the figure. (awarded to

REFERENCES 1.

Chen, S.H., Habib, G., Yang, C.Y., Gu, Z.W., Lee, B.R., Weng, S.A., Silberman,S.R., Cai, S.J., Deslypere, J.P., Rosseneu, M., Gotto, A.M. Li, W.H. and Chan, L. (1987) Science 238:36:3-366. 2. Powell, L.M., Wallis, S.C., Pease, R.J., Edwards, Y.H., Knott, T.J. and Scott, J. (1987) Cell 50:831-840. 3. Higuchi, K., Hospattankar, A.V., Law, SW., Meglin, N. Cortright, J. and Brewer, H.B. (1988) Proc. Natl. Acad. Sci. USA 851772-1776. 4. Gualberto, J.M., Lamattina, L., Bonnard, G., Weil, J.H. and Grienenberger, J.M. (1989) Nature 341:660-662. 5. Feagin, J.E., Abraham, J.M. and Stuart, K. (1988) Cell 53:413-422. 6. Cattaneo, R., Kaelin, K., Baczko, K. and Billeter, M.A. (1989) Cell 56:759-764. 7. Sparks, C.E., Hnatiuk, 0. and Marsh, J.B. (1981) Can. J. Biochem. 59:693-69. 8. Demmer, L.A., Levin, MS., Elovson, J., Reuben, M.A., Lusis, A.J. and Gordon, J.I. (1986) Proc. Natl. Acad. Sci. USA 83:8102-8106. 9. Imaizurni, K., Lu, Y.F. and Sugano, M. (1985) Biochim. Biophys. Acta 837:345-348. 10. Coleman, R.A., Haynes, E.B., Sand, T.M. and Davis, R.A. (1988) J. Lipid Res. 29:33-42. 517

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170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Davidson, N.O., Powell, L.M., Wallis, SC. and Scott, J. (1988) J. Biol. Chem. 263:1348213485. Davidson, N.O., Carlos, R.C., Drewek, M.J. and Parmer, T.G. (1988) J. Lipid Res. 29:151 l1522. Sparks, C.E., and Marsh, J.B. (1981) J. Lipid Res. 22:519-527. Elovson, J., Huang, Y.O., Baker, N. and Kannan, R. (1981) Proc. Natl. Acad. Sci. USA 77: 156-I 61. Rash, J.M., Rothblat, G.H. and Sparks, C.E. (1981) Biochim. Biophys Acta 666:294-298. Edge, S.B., Hoeg, J.M., Schneider, P.D. and Brewer, H.B. (1985) Metabolism 34:726-730. Gibbs, R.A., Nguyen, P.N. and Caskey, C.T. (1989) Nucleic Acids Res. 17:2437-2448. PCR Technology (1989) (Erlich, H.A. ed.) M Stockton Press, NY. Sparks, J.D. and Sparks, C.E. (1990) J. Biol. Chem. 265:ln press. Davidson, N.O., Teng, B. and Verp, M. (1990) Clin Res. 38:444A.

518

Quantitation of endogenous liver apolipoprotein B mRNA editing.

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein dependin...
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