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
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