Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 74-80
November 30, 1990
A P O L I P O P R O T E I N B M E S S E N G E R R N A E D I T I N G IS D E V E L O P M E N T A L L Y R E G U L A T E D IN PIG SMALL INTESTINE: N U C L E O T I D E C O M P A R I S O N OF A P O L I P O P R O T E I N B E D I T I N G REGIONS IN FIVE SPECIES
BaBie
Teng*,
Dennis
D. Black#
and Nicholas
O. D a v i d s o n *,I
Gastroenterology Section,*Departments of M e d i c i n e and #Pediatrics U n i v e r s i t y of Chicago,5841 South M a r y l a n d Avenue Chicago, Illinois 60637 Received September 13, 1990 A p o l i p o p r 0 t e i n B (apo B) m R N A undergoes a p o s t t r a n s c r i p t i o n a l tissue-specific e d i t i n g reaction which changes codon 2153 from g l u t a m i n e (CAA) in apoB-100 m R N A to an in-frame stop codon (UAA) in apoB-48 mRNA. Small intestinal apo B m R N A was found to be predominantly (>90%) u n e d i t e d in fetal (40-day g e s t a t i o n a l age) p i g l e t s but >95% e d i t e d in neonatal, suckling and adult animals. By contrast, b o t h fetal, neonatal and adult p i g liver c o n t a i n e d >99% unedited, apo B-100 mRNA. The n u c l e o t i d e sequence s p a n n i n g the e d i t e d region in apo B m R N A was found to be h i g h l y conserved. We s p e c u l a t e that the regulation of apo B m R N A e d i t i n g may be d e v e l o p m e n t a l l y m o d u l a t e d in pig small intestine. @1990Academic Press, Inc.
Apolipoprotein
B
of t r i g l y c e r i d e - r i c h apo B exists
which
contains
arises
(5,6)
lipogenesis suggests in rat
demonstrated in human
accounts
amino
mechanism
(CAA)
whereas acids
changes
to an in-frame
liver,
as apo B-
in the apo B-48,
of apo B-100, in the
codon
small
2153
translational
(3,4) .
(7) and t h y r o i d
We and others
that
and liver
status
intestine
(9).
(i0).
iTo whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
synthesizes shown
74
that
synthesis
in apo stop both apo B
in rat
in h e p a t i c (5,8).
Recent
is also d e v e l o p m e n t a l l y
apo B m R N A editing
small
have
of apoB
to a l t e r a t i o n s
hormone
apo B m R N A editing intestine
fetal
the rat liver
for this p a t t e r n
and is responsive
that
small
is s y n t h e s i z e d acids
form of RNA e d i t i n g
editing
with human
and apo B-48
mRNA editing liver
2152
Plasma
scale
in apo B-48 mRNA.
By contrast apo B-100
This
from glutamine
(UAA)
apo B-100
component
(LDL).
on a centile
of 4563 amino
of a novel
structural
lipoprotein
classified
the a m i n o - t e r m i n a l
(1,2).
mRNA
codon
protein
as a result
intestine B-100
forms,
In humans,
liver as a m a t u r e
is an e s s e n t i a l
and low d e n s i t y
in two
i00 and apo B-48.
(apo B)
We have
work regulated
recently
is d e v e l o p m e n t a l l y Furthermore,
regulated
the p r o g r e s s i v e
Vol. 173, No. 1, 1990
increase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in apo B m R N A e d i t i n g
pattern
of apo B synthesis
studies
were
neonatal
undertaken
and adult
demonstrates
many
pig
to examine small
(11,12).
Materials
and
since
to humans
with a c o r r e s p o n d i n g
(I0).
apo B m R N A
intestine
similarities
metabolism
was a s s o c i a t e d
and secretion
The
current
editing this
in fetal,
animal
in regard
to l i p o p r o t e i n
Methods
Tissues: Small i n t e s t i n e and liver of forty day g e s t a t i o n a l age (fetal), 2 day (newborn), 1-2 week (suckling), and adult (maternal) female d o m e s t i c swine were s u r g i c a l l y r e m o v e d and w a s h e d e x t e n s i v e l y in ice-cold sterile saline. P o r t i o n s were s n a p - f r o z e n in l i q u i d - n i t r o g e n and R N A e x t r a c t e d as d e s c r i b e d (10,13). Human intestinal and h e p a t i c R N A was p r e p a r e d as p r e v i o u s l y d e s c r i b e d (i0). P r i m e r e x t e n s i o n assay: A n a l y s i s of apo B m R N A editing was p e r f o r m e d on total RNA with m o d i f i c a t i o n s to the m e t h o d of D r i s c o l l et al (14). Briefly, total R N A (5ug) was d e n a t u r e d at 75 degrees C for I0 m i n u t e s and a n n e a l e d to 3 2 p - e n d l a b e l e d o l i g o n u c l e o t i d e BTI(5'AATCATGTAAATCATAACTATCTTTAATATACTGA, 5'end at 6674, c o m p l e m e n t a r y to human apoB cDNA) at 45 degrees C overnight. The a n n e a l e d p r o d u c t s were e x t e n d e d in the p r e s e n c e of 500uM e a c h dATP, dCTP, dTTP and d i d e o x y GTP (Pharmacia), using I0 units of M M L V reverse t r a n s c r i p t a s e (BRL) at 42 degrees C for 90 minutes. P r o d u c t s were a n a l y z e d by 8% p 0 1 y a c r y l a m i d e - u r e a gel e l e c t r o p h o r e s i s and s u b j e c t e d to a u t o r a d i o g r a p h y at -70 degrees C. The ratio of e d i t e d to u n e d i t e d apo B m R N A was d e t e r m i n e d by laser d e n s i t o m e t r i c scanning. ADoB DNA S e a u e n c e F l a n k i n Q the E d i t e d Base: A 514 b a s e p a i r (bp) fragment flanking n u c l e o t i d e 6666 in apo B c D N A from n u c l e o t i d e s 6272 to 6786 was a m p l i f i e d from p i g and mouse g e n o m i c D N A using Taq DNA p o l y m e r a s e w i t h h u m a n p r i m e r s PCR 12 ( 5 ' G C C C C A A G A A T T T A C A A T T G T T G C , 5'end at n u c l e o t i d e 6272) and PCR i0 (5'CACGGATATGATAGTGCTCAT, 5'end at n u c l e o t i d e 6786). The a m p l i f i e d fragment was then cloned into MI3 [vector mpl9] for DNA sequencing. The sequence i n f o r m a t i o n was c o n f i r m e d by repeat analysis u s i n g a c o m p l e m e n t a r y h o m o l o g o u s internal p r i m e r (5'CAAGAATTTTCAATGTTGC, 5' end at 6765). Intestinal apo B cDNA f l a n k i n g the e d i t e d b a s ~ w a s also s e q u e n c e d f o l l o w i n g reverse t r a n s c r i p t i o n and p o l y m e r a s e chain reaction a m p l i f i c a t i o n of total RNA. S e q u e n c i n g was c a r r i e d out by dideoxy chain t e r m i n a t i o n (15) using b o t h K l e n o w and sequenase methods (sequenase D N A s e q u e n c i n g kit, U n i t e d B i o c h e m i c a l Corporation). N u c l e o t i d e s e q u e n c e s were a n a l y z e d u s i n g the B e c k m a n M i c r o g e n i e program. Results
Apo B m R N A intestine latter
editing
and liver
animals
fat-enriched
studied
diets
extension
assay,
the
upstream
first
nucleotide
was
analyzed
from fetal, following
(Fig i).
the p r i m e r cytidine,
6666 of apo B-100
in samples
maternal
consumption
Under
located
75
animals,
of either
the c o n d i t i o n s
anneals mRNA
of pig small
and n e w b o r n
low-fat
or
of the p r i m e r
to apo B m R N A and extends at n u c l e o t i d e
remains
the
unedited,
6666.
to
If
a product
of
Vol.
173,
No.
1, 1 9 9 0
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pig
Human I
Adult L
I
M I
F
L
I
L
I
NB
NB
VlV
FAT L I
L
I
Suckling FAT L I
- UAA
-CAA - Primer
Figure
I.
Developmental regulation of pig intestinal apo B mRNA editing determined by RNA primer extension. Total RNA from pig liver (L) and small intestine (I) of maternal (M), 40 day fetal (F), newborn (NB) animals-the latter fed either a low-fat (VIVONEX, VIV) or fat-rich diet (FAT) - and suckling animals were used to quantitate unedited (CAA) and edited (UAA) apo B mRNA. Total RNA from human adult liver (L) and small intestine (I) was assayed in parallel and shown for comparison. A representative example of 2 individual assays, each conducted in duplicate, is shown. 43 bases
is p r o d u c e d
to a U, the p r i m e r
(CAA)
6655 with an e x t e n s i o n identical
biosynthetically
(UAA)
transcript
shown that adult
intestinal
nucleotide
nucleotides
nucleotides
By contrast, fetal,
apo B m R N A
(CAA) .
by direct 6565-6765
there
newborn,
which
or
The
sequencing
D N A with the e x c e p t i o n
of the D N A a n d t r a n s l a t e d
the apo B m R N A e d i t i n g
6408-6765
sequences
and residues
2092-2185
of
showed
of
and part
of the m o u s e (1,5,16,17). (Table
in this sequences This
region.
acid
of h u m a n
apoB
rabbit,
sequence
The human,
to
cDNA rat,
to rabbit,
have been p r e v i o u s l y
region
is h i g h l y
i) and the h o m o l o g y 76
amino
- corresponding
The pig,
are a l i g n e d w i t h the h u m a n
the c o n s e r v a t i o n
five species
region
2A and 2B r e s p e c t i v e l y .
characterized the
we have p r e v i o u s l y
(12).
were c o n f i r m e d
to g e n o m i c
comparison
in F i g u r e s
illustrate rat,
extension
edited
in the n e w b o r n
from e i t h e r
>99% u n e d i t e d
setting
and
predominantly
setting
is s y n t h e s i z e d
in this
suckling,
at
to
6666.
spanning
and m o u s e
In this
was
intestine
Attempts
in-vivo,
Newborn,
edited transcript
sequence
A species
is shown
fat.
small
examined.
with no d i f f e r e n c e s
apo B CDNA b e t w e e n
an i d e n t i c a l
sequence
(95%)
all c o n t a i n i n g
of p r i m e r
in fetal p i g
shown).
conditions
Apo B m R N A e d i t i n g
apoB m R N A all c o n t a i n e d
only apo B-48
liver,
results
(data not
to d i e t a r y
was no d e t e c t a b l e
8% UAA)
6666 is e d i t e d
C at n u c l e o t i d e
(UAA),
i) .
label the apo B species,
intestinal
attributable
(Fig.
the only t i m e - p o i n t
were u n s u c c e s s f u l maternal
of 54 bases
and p i g
(approximately
40 days gestation,
if n u c l e o t i d e
to the next u p s t r e a m
product
in b o t h h u m a n
detectable
whereas,
extends
conserved
is g r e a t e r
among
at the
Vol. 173, No. 1, 1990
A
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Human Pig Rabbit Rat Mouse
6482 6522 CCCACAGCAAGCTAATGATTATCTGAATTCATTCAATTGGGAGAGACAAGTTTCACATGC ........... T .............. G-A ...... G ...... A .......... TGAG--........... T .............. G-A ......................... CAG--T--T ..... GAT-C .............. G---CTG-C .............. AG-TGG--T--T ..... GAT-C--C ........... G---CTG-C .............. AG-TGG---
Human Pig Rabbit Rat Mouse
6542 6582 CAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAATTACAGAAAATGATATACAAAT ---AA ...... AT-G--A-T---TGG--G-T ..................... G---G--............ A---A--T .......... T .... A ......... G ............. C ...... A---T-A---T--T---TGG .... C .............. T ...... G---T--...... A---A-A---T--T---TGG .... T .............. T ...... G---T---
Human Pig
6602 6642 TGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTGCAGACATATAT ......... CA .............. C .............. A .... G--A--A
Rabbit Rat Mouse
...... G---A ................ ---C ...... AG ............... ---CA ..... AG ...............
Human Pig Rabbit Rat Mouse
6662 6702 GATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTAT ............................. A .................. T--G-C ...... ............................. A--T .... C .......... T ........... ....................... G ..... A ....... GC---G--C--A .... G-A .... - ............................ A ....... CC ...... C--A .... G-A ....
Human Pig Rabbit Rat Mouse
6722 TGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTG ..... GG .................... C--C---G .... A .... G ...... A---C ....... G ............. .... C-G ......... AG ............ GC ...... .... G-G ......... CG ............ G .......
Figure
nucleotide species.
apoB-100 on
top
in
of
corresponds
and
the
The
the
four
apo
complete
nucleotide
other
represent
nucleotides
sequences numbering (16).
line,
sequences The
The
sequence
the
Hyphens
G ........ C ........ C ........
......
G-
T ......... GTG ....... CGC TG ....... CGC
6762 T ..... T ..... TG .... TG ....
2A.
Aligned five
A ........... G ........... C ...........
B
mRNA to
human
differences
species
nucleotides corresponding
to
the
to apo
is
human
the
are mRNA
provided
aligned
human
in
human
from the
B
regions
published
sequence
compared
identical
editing
the
beneath.
sequence.
editing
codon
is
highlighted.
B Human Pig Rabbit Rat Mouse
2092 2132 PQQANDYLNSFNWERQVSHAKEKLTALTKKYRITENDIQIALDDAKINFNEKLSQLQTYM ---V .... ST-S ..... LS--K-HSDFMED ....... VR .... N .... L .... T ..... V ---V .... ST ........ S ...... TF--N-K ....... T---N .... L .......... V ---IH .... ASD ..... AG ...... SFMEN .... D--VL .... S .... L ....... E--A ---IHH---ASD ..... AG .... I-SFMEN .... D--VL--I-S ............ E--A
Human Pig
2152 2182 IQFDQYIKDSYDLHDLKIAIANIIDEIIEKLKSL ......... N ..... F-T---R ...... AT--I-
Rabbit Rat Mouse
......... NF .... F ..... S---Q-M .... I....... R-N--AQ---RT--Q---R ...... M......... N--P .... RT--E---R ...... M-
Figure
2B.
Aligned
amino
species. i00
sequence
provided indicated to
the
acid
The
sequences
numbering (16).
on
the
below. human
The
top
line,
Hyphens
sequence.
of
apo
corresponds complete and
mRNA
human amino
represent The
B to
single
employed.
77
editing
the amino
acid
regions
published acid
residue
in
human sequence
acid
residues
letter
code
for
amino
B-
is
differences
amino
five
apo
are
identical acids
is
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Nucleotide Homology Surrounding the Apo B mRNA Editing Regions of Five Species
Human
Pig
Rabbit
Rat
Mouse
-
85.6
89.4
80.1
80.6
85.2
77.3
77.8
79.7
81.3
Human Pig
85.6
-
Rabbit
89.4
88.2
Rat
80.1
77.3
79.7
Mouse
80.6
77.8
81.3
-
94.4 94.4
The comparisons are expressed as percent homology between pairs of sequences. The homologies between nucleotide 6408-6765 were calculated using the Microgenie Compare/Align program. n u c l e o t i d e t h a n at the amino a c i d level. h o m o l o g y was h i g h e r b e t w e e n human, human,
rat a n d mouse.
rat a n d m o u s e
However,
pig,
The o v e r a l l n u c l e o t i d e and r a b b i t t h a n b e t w e e n
the n u c l e o t i d e h o m o l o g y b e t w e e n
sequences exceeded
94%.
The m i n i m u m r e q u i s i t e
s e q u e n c e of 26bp r e q u i r e d for apo B m R N A e d i t i n g
in v i t r o
identical
species and
appears
in the pig to that d e s c r i b e d
enriched
in a d e n o s i n e
r e g i o n s of apo B m R N A
for o t h e r
and u r i d i n e
is
c o m p a r e d to o t h e r
(17).
Discussion
This
study d e m o n s t r a t e s that p i g i n t e s t i n a l apo B-48
g e n e r a t e d by a c o n s e r v e d R N A e d i t i n g m e c h a n i s m . relatively inactive detectable
in fetal
small
intestine,
in any s a m p l e of h e p a t i c RNA.
is
This p r o c e s s
is
but was not
Although newborn piglets
were f o u n d to h a v e an i n d i s t i n g u i s h a b l e p a t t e r n of i n t e s t i n a l apo B m R N A e d i t i n g to that the d e v e l o p m e n t a l
found in adult animals,
switch remains uncertain.
the e x a c t t i m i n g of
Additionally,
it
r e m a i n s u n c l e a r as to w h e t h e r i n t e s t i n a l apo B - 1 0 0 m R N A is actively translated fetal
small
increase synthesis of h u m a n
in the
intestinal
fetal pig.
Our r e c e n t
in e d i t i n g d u r i n g the s e c o n d t r i m e s t e r , and s e c r e t i o n of b o t h apo B i s o f o r m s fetal
small
recent observations
of h u m a n
with active (i0) .
The t i m i n g
i n t e s t i n a l apo B m R N A e d i t i n g c o n t r a s t s in the rat where small
e d i t i n g r e a c h e s adult Furthermore,
studies
apo B m R N A e d i t i n g s u g g e s t a p r o g r e s s i v e
levels
with
i n t e s t i n a l apo B m R N A
just p r i o r to b i r t h
(9).
rat h e p a t i c apo B m R N A e d i t i n g d e m o n s t r a t e d a
developmental profile a c h i e v i n g adult
levels
distinct
f r o m e i t h e r of t h e s e p a t t e r n s ,
24 days a f t e r b i r t h 78
(9).
The e v i d e n c e thus
Vol. 173, No. 1, 1990
points
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to the existence
specific
factors
of both
species-specific
in the developmental
and tissue-
regulation
of apo B mRNA
editing. Although
the molecular
developmentally observations
regulated
point
hormonal
factors
temporal
emergence
to the functional organ.
However,
mechanisms
process
to the likely
(5,7-8).
of intestinal commitment
the nature
of nucleotide
requirements studies,
gut remains
sequence
a functional
site-specific
relatively
lax sequence
nucleotide
6666
(18).
for apo B mRNA editing
and
that the
intestine
advantage unclear.
of apo B The
of the stringency On the other hand recent
mutagenesis,
requirement
as a lipogenic
spanning the RNA editing
definition
for apo B mRNA editing.
using
our recent
of both metabolic
apo B mRNA editing may be related
of the presumed
conservation
this
we speculate
of the small
in the developing
implies
remain unknown, involvement
In particular,
mRNA editing region
underlying
have demonstrated
immediately
Thus the precise
a
flanking
structural
requirements
have yet to be defined.
Acknowledgments We would like to thank Drs. V. Sukhatme and G.I. Bell for advice and Ms. C. Sullivan for outstanding secretarial assistance. Supported by NIH grants DK26678 (BT),HL38180,HL02166 (NOD),R29HD22551 (DDB),AHA Grant-In-Aid #88-1015 and Digestive Disease Research Center Grant DK42086 (NOD).
References i.
Powell, L.M., Wallis, S.C., Pease, R.J., Edwards, Y.H., Knott, T.J. and Scott, J. (1987) Cell 50,831-840. 2. Chen, S-H., Habib, G., Yang, C-Y., Gu, X-W., Lee, B.R., Weng, S-A., Silberman, S.R., Cai, S-J., Deslypere, J.P., Rosseneu, M., Gotto, A.M., Jr., Li, W-H. and Chan, L. (1987) Science 238, 362-366. 3. Krishnaiah, K.V., Walker, L.F., Borensztajn, J., Schonfeld G. and Getz, G.S. (1980) Proc. Natl. Acad. Sci. USA 77, 3806-3810. 4. Elovson, J., Huang, Y.O. Baker, N. and Kannan, R. (1981) Proc. Natl. Acad. Sci. USA 78, 157-161. 5. Davidson, N.O., Powell, L.M., Wallis, S.C. and Scott, J. (1988) J. Biol. Chem. 263, 13482-13485. 6. Tennyson, G.E., Sabatos, C.A., Higuchi, K., Meglin, N. and Brewer, H.B., Jr. (1989) Proc. Natl. Acad. Sci. USA 86, 500504. 7. Baum, C., Teng, B-B. and Davidson, N.O. (1990) J. Biol. Chem. In press. 8. Davidson, N.O., Carlos, R.C. and Lukascewicz, A.M. (1990) Mol. Endocrinol. 4, 779-785. 9. Wu, J.H., Semenkovich, C.F., Chen, S-H., Li, W-H. and Chan, L. (1990) J. Biol. Chem. 265, 12312-12316. I0. Teng, B-B., Verp, M., Salomon, J. and Davidson, N.O. (1990) J. Biol. Chem. In press. ii. Chapman, M.J. (1986) Methods Enzymol. 128, 70-143. 79
VoI. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12. Black, D.D. and Davidson, N.O. (1989) J. Lipid Res. 30, 207218. 13. Chomczynski, P. and Sacehi, N. (1987) Anal. Biochem. 162, 156159. 14. Driscoll, D.M., Wynne, J.K., Wallis, S.C. and Scott, J. (1989) Cell 58, 519-525. 15. Sanger, F. and Coulson, A.R. (1978) FEBS Lett. 87, 107-110. 16. Knott, T.J., Wallis, S.C., Powell, L.M., Pease, R.J., Lusis, A.J., Blackhart, B., McCarthy, B.J., Mahley, R.W., Levy-Wilson, B. and Scott, J. (1986) Nucleic Acids Res. 14, 7501-7503. 17. Davies, M.S., Wallis, S.C., Driscoll, D.M., Wynne, J.K., Williams, G.W., Powell, L.M. and Scott, J. (1989) J. Biol. Chem. 264, 13395-13398. 18. Chen, S-H., Li, X., Liao, W.S.L., Wu, J.H. and Chan, L. (1990) J. Biol. Chem. 265, 6811-6816.
80
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 74-80
November 30, 1990
A P O L I P O P R O T E I N B M E S S E N G E R R N A E D I T I N G IS D E V E L O P M E N T A L L Y R E G U L A T E D IN PIG SMALL INTESTINE: N U C L E O T I D E C O M P A R I S O N OF A P O L I P O P R O T E I N B E D I T I N G REGIONS IN FIVE SPECIES
BaBie
Teng*,
Dennis
D. Black#
and Nicholas
O. D a v i d s o n *,I
Gastroenterology Section,*Departments of M e d i c i n e and #Pediatrics U n i v e r s i t y of Chicago,5841 South M a r y l a n d Avenue Chicago, Illinois 60637 Received September 13, 1990 A p o l i p o p r 0 t e i n B (apo B) m R N A undergoes a p o s t t r a n s c r i p t i o n a l tissue-specific e d i t i n g reaction which changes codon 2153 from g l u t a m i n e (CAA) in apoB-100 m R N A to an in-frame stop codon (UAA) in apoB-48 mRNA. Small intestinal apo B m R N A was found to be predominantly (>90%) u n e d i t e d in fetal (40-day g e s t a t i o n a l age) p i g l e t s but >95% e d i t e d in neonatal, suckling and adult animals. By contrast, b o t h fetal, neonatal and adult p i g liver c o n t a i n e d >99% unedited, apo B-100 mRNA. The n u c l e o t i d e sequence s p a n n i n g the e d i t e d region in apo B m R N A was found to be h i g h l y conserved. We s p e c u l a t e that the regulation of apo B m R N A e d i t i n g may be d e v e l o p m e n t a l l y m o d u l a t e d in pig small intestine. @1990Academic Press, Inc.
Apolipoprotein
B
of t r i g l y c e r i d e - r i c h apo B exists
which
contains
arises
(5,6)
lipogenesis suggests in rat
demonstrated in human
accounts
amino
mechanism
(CAA)
whereas acids
changes
to an in-frame
liver,
as apo B-
in the apo B-48,
of apo B-100, in the
codon
small
2153
translational
(3,4) .
(7) and t h y r o i d
We and others
that
and liver
status
intestine
(9).
(i0).
iTo whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
synthesizes shown
74
that
synthesis
in apo stop both apo B
in rat
in h e p a t i c (5,8).
Recent
is also d e v e l o p m e n t a l l y
apo B m R N A editing
small
have
of apoB
to a l t e r a t i o n s
hormone
apo B m R N A editing intestine
fetal
the rat liver
for this p a t t e r n
and is responsive
that
small
is s y n t h e s i z e d acids
form of RNA e d i t i n g
editing
with human
and apo B-48
mRNA editing liver
2152
Plasma
scale
in apo B-48 mRNA.
By contrast apo B-100
This
from glutamine
(UAA)
apo B-100
component
(LDL).
on a centile
of 4563 amino
of a novel
structural
lipoprotein
classified
the a m i n o - t e r m i n a l
(1,2).
mRNA
codon
protein
as a result
intestine B-100
forms,
In humans,
liver as a m a t u r e
is an e s s e n t i a l
and low d e n s i t y
in two
i00 and apo B-48.
(apo B)
We have
work regulated
recently
is d e v e l o p m e n t a l l y Furthermore,
regulated
the p r o g r e s s i v e
Vol. 173, No. 1, 1990
increase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in apo B m R N A e d i t i n g
pattern
of apo B synthesis
studies
were
neonatal
undertaken
and adult
demonstrates
many
pig
to examine small
(11,12).
Materials
and
since
to humans
with a c o r r e s p o n d i n g
(I0).
apo B m R N A
intestine
similarities
metabolism
was a s s o c i a t e d
and secretion
The
current
editing this
in fetal,
animal
in regard
to l i p o p r o t e i n
Methods
Tissues: Small i n t e s t i n e and liver of forty day g e s t a t i o n a l age (fetal), 2 day (newborn), 1-2 week (suckling), and adult (maternal) female d o m e s t i c swine were s u r g i c a l l y r e m o v e d and w a s h e d e x t e n s i v e l y in ice-cold sterile saline. P o r t i o n s were s n a p - f r o z e n in l i q u i d - n i t r o g e n and R N A e x t r a c t e d as d e s c r i b e d (10,13). Human intestinal and h e p a t i c R N A was p r e p a r e d as p r e v i o u s l y d e s c r i b e d (i0). P r i m e r e x t e n s i o n assay: A n a l y s i s of apo B m R N A editing was p e r f o r m e d on total RNA with m o d i f i c a t i o n s to the m e t h o d of D r i s c o l l et al (14). Briefly, total R N A (5ug) was d e n a t u r e d at 75 degrees C for I0 m i n u t e s and a n n e a l e d to 3 2 p - e n d l a b e l e d o l i g o n u c l e o t i d e BTI(5'AATCATGTAAATCATAACTATCTTTAATATACTGA, 5'end at 6674, c o m p l e m e n t a r y to human apoB cDNA) at 45 degrees C overnight. The a n n e a l e d p r o d u c t s were e x t e n d e d in the p r e s e n c e of 500uM e a c h dATP, dCTP, dTTP and d i d e o x y GTP (Pharmacia), using I0 units of M M L V reverse t r a n s c r i p t a s e (BRL) at 42 degrees C for 90 minutes. P r o d u c t s were a n a l y z e d by 8% p 0 1 y a c r y l a m i d e - u r e a gel e l e c t r o p h o r e s i s and s u b j e c t e d to a u t o r a d i o g r a p h y at -70 degrees C. The ratio of e d i t e d to u n e d i t e d apo B m R N A was d e t e r m i n e d by laser d e n s i t o m e t r i c scanning. ADoB DNA S e a u e n c e F l a n k i n Q the E d i t e d Base: A 514 b a s e p a i r (bp) fragment flanking n u c l e o t i d e 6666 in apo B c D N A from n u c l e o t i d e s 6272 to 6786 was a m p l i f i e d from p i g and mouse g e n o m i c D N A using Taq DNA p o l y m e r a s e w i t h h u m a n p r i m e r s PCR 12 ( 5 ' G C C C C A A G A A T T T A C A A T T G T T G C , 5'end at n u c l e o t i d e 6272) and PCR i0 (5'CACGGATATGATAGTGCTCAT, 5'end at n u c l e o t i d e 6786). The a m p l i f i e d fragment was then cloned into MI3 [vector mpl9] for DNA sequencing. The sequence i n f o r m a t i o n was c o n f i r m e d by repeat analysis u s i n g a c o m p l e m e n t a r y h o m o l o g o u s internal p r i m e r (5'CAAGAATTTTCAATGTTGC, 5' end at 6765). Intestinal apo B cDNA f l a n k i n g the e d i t e d b a s ~ w a s also s e q u e n c e d f o l l o w i n g reverse t r a n s c r i p t i o n and p o l y m e r a s e chain reaction a m p l i f i c a t i o n of total RNA. S e q u e n c i n g was c a r r i e d out by dideoxy chain t e r m i n a t i o n (15) using b o t h K l e n o w and sequenase methods (sequenase D N A s e q u e n c i n g kit, U n i t e d B i o c h e m i c a l Corporation). N u c l e o t i d e s e q u e n c e s were a n a l y z e d u s i n g the B e c k m a n M i c r o g e n i e program. Results
Apo B m R N A intestine latter
editing
and liver
animals
fat-enriched
studied
diets
extension
assay,
the
upstream
first
nucleotide
was
analyzed
from fetal, following
(Fig i).
the p r i m e r cytidine,
6666 of apo B-100
in samples
maternal
consumption
Under
located
75
animals,
of either
the c o n d i t i o n s
anneals mRNA
of pig small
and n e w b o r n
low-fat
or
of the p r i m e r
to apo B m R N A and extends at n u c l e o t i d e
remains
the
unedited,
6666.
to
If
a product
of
Vol.
173,
No.
1, 1 9 9 0
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pig
Human I
Adult L
I
M I
F
L
I
L
I
NB
NB
VlV
FAT L I
L
I
Suckling FAT L I
- UAA
-CAA - Primer
Figure
I.
Developmental regulation of pig intestinal apo B mRNA editing determined by RNA primer extension. Total RNA from pig liver (L) and small intestine (I) of maternal (M), 40 day fetal (F), newborn (NB) animals-the latter fed either a low-fat (VIVONEX, VIV) or fat-rich diet (FAT) - and suckling animals were used to quantitate unedited (CAA) and edited (UAA) apo B mRNA. Total RNA from human adult liver (L) and small intestine (I) was assayed in parallel and shown for comparison. A representative example of 2 individual assays, each conducted in duplicate, is shown. 43 bases
is p r o d u c e d
to a U, the p r i m e r
(CAA)
6655 with an e x t e n s i o n identical
biosynthetically
(UAA)
transcript
shown that adult
intestinal
nucleotide
nucleotides
nucleotides
By contrast, fetal,
apo B m R N A
(CAA) .
by direct 6565-6765
there
newborn,
which
or
The
sequencing
D N A with the e x c e p t i o n
of the D N A a n d t r a n s l a t e d
the apo B m R N A e d i t i n g
6408-6765
sequences
and residues
2092-2185
of
showed
of
and part
of the m o u s e (1,5,16,17). (Table
in this sequences This
region.
acid
of h u m a n
apoB
rabbit,
sequence
The human,
to
cDNA rat,
to rabbit,
have been p r e v i o u s l y
region
is h i g h l y
i) and the h o m o l o g y 76
amino
- corresponding
The pig,
are a l i g n e d w i t h the h u m a n
the c o n s e r v a t i o n
five species
region
2A and 2B r e s p e c t i v e l y .
characterized the
we have p r e v i o u s l y
(12).
were c o n f i r m e d
to g e n o m i c
comparison
in F i g u r e s
illustrate rat,
extension
edited
in the n e w b o r n
from e i t h e r
>99% u n e d i t e d
setting
and
predominantly
setting
is s y n t h e s i z e d
in this
suckling,
at
to
6666.
spanning
and m o u s e
In this
was
intestine
Attempts
in-vivo,
Newborn,
edited transcript
sequence
A species
is shown
fat.
small
examined.
with no d i f f e r e n c e s
apo B CDNA b e t w e e n
an i d e n t i c a l
sequence
(95%)
all c o n t a i n i n g
of p r i m e r
in fetal p i g
shown).
conditions
Apo B m R N A e d i t i n g
apoB m R N A all c o n t a i n e d
only apo B-48
liver,
results
(data not
to d i e t a r y
was no d e t e c t a b l e
8% UAA)
6666 is e d i t e d
C at n u c l e o t i d e
(UAA),
i) .
label the apo B species,
intestinal
attributable
(Fig.
the only t i m e - p o i n t
were u n s u c c e s s f u l maternal
of 54 bases
and p i g
(approximately
40 days gestation,
if n u c l e o t i d e
to the next u p s t r e a m
product
in b o t h h u m a n
detectable
whereas,
extends
conserved
is g r e a t e r
among
at the
Vol. 173, No. 1, 1990
A
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Human Pig Rabbit Rat Mouse
6482 6522 CCCACAGCAAGCTAATGATTATCTGAATTCATTCAATTGGGAGAGACAAGTTTCACATGC ........... T .............. G-A ...... G ...... A .......... TGAG--........... T .............. G-A ......................... CAG--T--T ..... GAT-C .............. G---CTG-C .............. AG-TGG--T--T ..... GAT-C--C ........... G---CTG-C .............. AG-TGG---
Human Pig Rabbit Rat Mouse
6542 6582 CAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAATTACAGAAAATGATATACAAAT ---AA ...... AT-G--A-T---TGG--G-T ..................... G---G--............ A---A--T .......... T .... A ......... G ............. C ...... A---T-A---T--T---TGG .... C .............. T ...... G---T--...... A---A-A---T--T---TGG .... T .............. T ...... G---T---
Human Pig
6602 6642 TGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTGCAGACATATAT ......... CA .............. C .............. A .... G--A--A
Rabbit Rat Mouse
...... G---A ................ ---C ...... AG ............... ---CA ..... AG ...............
Human Pig Rabbit Rat Mouse
6662 6702 GATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTAT ............................. A .................. T--G-C ...... ............................. A--T .... C .......... T ........... ....................... G ..... A ....... GC---G--C--A .... G-A .... - ............................ A ....... CC ...... C--A .... G-A ....
Human Pig Rabbit Rat Mouse
6722 TGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTG ..... GG .................... C--C---G .... A .... G ...... A---C ....... G ............. .... C-G ......... AG ............ GC ...... .... G-G ......... CG ............ G .......
Figure
nucleotide species.
apoB-100 on
top
in
of
corresponds
and
the
The
the
four
apo
complete
nucleotide
other
represent
nucleotides
sequences numbering (16).
line,
sequences The
The
sequence
the
Hyphens
G ........ C ........ C ........
......
G-
T ......... GTG ....... CGC TG ....... CGC
6762 T ..... T ..... TG .... TG ....
2A.
Aligned five
A ........... G ........... C ...........
B
mRNA to
human
differences
species
nucleotides corresponding
to
the
to apo
is
human
the
are mRNA
provided
aligned
human
in
human
from the
B
regions
published
sequence
compared
identical
editing
the
beneath.
sequence.
editing
codon
is
highlighted.
B Human Pig Rabbit Rat Mouse
2092 2132 PQQANDYLNSFNWERQVSHAKEKLTALTKKYRITENDIQIALDDAKINFNEKLSQLQTYM ---V .... ST-S ..... LS--K-HSDFMED ....... VR .... N .... L .... T ..... V ---V .... ST ........ S ...... TF--N-K ....... T---N .... L .......... V ---IH .... ASD ..... AG ...... SFMEN .... D--VL .... S .... L ....... E--A ---IHH---ASD ..... AG .... I-SFMEN .... D--VL--I-S ............ E--A
Human Pig
2152 2182 IQFDQYIKDSYDLHDLKIAIANIIDEIIEKLKSL ......... N ..... F-T---R ...... AT--I-
Rabbit Rat Mouse
......... NF .... F ..... S---Q-M .... I....... R-N--AQ---RT--Q---R ...... M......... N--P .... RT--E---R ...... M-
Figure
2B.
Aligned
amino
species. i00
sequence
provided indicated to
the
acid
The
sequences
numbering (16).
on
the
below. human
The
top
line,
Hyphens
sequence.
of
apo
corresponds complete and
mRNA
human amino
represent The
B to
single
employed.
77
editing
the amino
acid
regions
published acid
residue
in
human sequence
acid
residues
letter
code
for
amino
B-
is
differences
amino
five
apo
are
identical acids
is
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Nucleotide Homology Surrounding the Apo B mRNA Editing Regions of Five Species
Human
Pig
Rabbit
Rat
Mouse
-
85.6
89.4
80.1
80.6
85.2
77.3
77.8
79.7
81.3
Human Pig
85.6
-
Rabbit
89.4
88.2
Rat
80.1
77.3
79.7
Mouse
80.6
77.8
81.3
-
94.4 94.4
The comparisons are expressed as percent homology between pairs of sequences. The homologies between nucleotide 6408-6765 were calculated using the Microgenie Compare/Align program. n u c l e o t i d e t h a n at the amino a c i d level. h o m o l o g y was h i g h e r b e t w e e n human, human,
rat a n d mouse.
rat a n d m o u s e
However,
pig,
The o v e r a l l n u c l e o t i d e and r a b b i t t h a n b e t w e e n
the n u c l e o t i d e h o m o l o g y b e t w e e n
sequences exceeded
94%.
The m i n i m u m r e q u i s i t e
s e q u e n c e of 26bp r e q u i r e d for apo B m R N A e d i t i n g
in v i t r o
identical
species and
appears
in the pig to that d e s c r i b e d
enriched
in a d e n o s i n e
r e g i o n s of apo B m R N A
for o t h e r
and u r i d i n e
is
c o m p a r e d to o t h e r
(17).
Discussion
This
study d e m o n s t r a t e s that p i g i n t e s t i n a l apo B-48
g e n e r a t e d by a c o n s e r v e d R N A e d i t i n g m e c h a n i s m . relatively inactive detectable
in fetal
small
intestine,
in any s a m p l e of h e p a t i c RNA.
is
This p r o c e s s
is
but was not
Although newborn piglets
were f o u n d to h a v e an i n d i s t i n g u i s h a b l e p a t t e r n of i n t e s t i n a l apo B m R N A e d i t i n g to that the d e v e l o p m e n t a l
found in adult animals,
switch remains uncertain.
the e x a c t t i m i n g of
Additionally,
it
r e m a i n s u n c l e a r as to w h e t h e r i n t e s t i n a l apo B - 1 0 0 m R N A is actively translated fetal
small
increase synthesis of h u m a n
in the
intestinal
fetal pig.
Our r e c e n t
in e d i t i n g d u r i n g the s e c o n d t r i m e s t e r , and s e c r e t i o n of b o t h apo B i s o f o r m s fetal
small
recent observations
of h u m a n
with active (i0) .
The t i m i n g
i n t e s t i n a l apo B m R N A e d i t i n g c o n t r a s t s in the rat where small
e d i t i n g r e a c h e s adult Furthermore,
studies
apo B m R N A e d i t i n g s u g g e s t a p r o g r e s s i v e
levels
with
i n t e s t i n a l apo B m R N A
just p r i o r to b i r t h
(9).
rat h e p a t i c apo B m R N A e d i t i n g d e m o n s t r a t e d a
developmental profile a c h i e v i n g adult
levels
distinct
f r o m e i t h e r of t h e s e p a t t e r n s ,
24 days a f t e r b i r t h 78
(9).
The e v i d e n c e thus
Vol. 173, No. 1, 1990
points
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to the existence
specific
factors
of both
species-specific
in the developmental
and tissue-
regulation
of apo B mRNA
editing. Although
the molecular
developmentally observations
regulated
point
hormonal
factors
temporal
emergence
to the functional organ.
However,
mechanisms
process
to the likely
(5,7-8).
of intestinal commitment
the nature
of nucleotide
requirements studies,
gut remains
sequence
a functional
site-specific
relatively
lax sequence
nucleotide
6666
(18).
for apo B mRNA editing
and
that the
intestine
advantage unclear.
of apo B The
of the stringency On the other hand recent
mutagenesis,
requirement
as a lipogenic
spanning the RNA editing
definition
for apo B mRNA editing.
using
our recent
of both metabolic
apo B mRNA editing may be related
of the presumed
conservation
this
we speculate
of the small
in the developing
implies
remain unknown, involvement
In particular,
mRNA editing region
underlying
have demonstrated
immediately
Thus the precise
a
flanking
structural
requirements
have yet to be defined.
Acknowledgments We would like to thank Drs. V. Sukhatme and G.I. Bell for advice and Ms. C. Sullivan for outstanding secretarial assistance. Supported by NIH grants DK26678 (BT),HL38180,HL02166 (NOD),R29HD22551 (DDB),AHA Grant-In-Aid #88-1015 and Digestive Disease Research Center Grant DK42086 (NOD).
References i.
Powell, L.M., Wallis, S.C., Pease, R.J., Edwards, Y.H., Knott, T.J. and Scott, J. (1987) Cell 50,831-840. 2. Chen, S-H., Habib, G., Yang, C-Y., Gu, X-W., Lee, B.R., Weng, S-A., Silberman, S.R., Cai, S-J., Deslypere, J.P., Rosseneu, M., Gotto, A.M., Jr., Li, W-H. and Chan, L. (1987) Science 238, 362-366. 3. Krishnaiah, K.V., Walker, L.F., Borensztajn, J., Schonfeld G. and Getz, G.S. (1980) Proc. Natl. Acad. Sci. USA 77, 3806-3810. 4. Elovson, J., Huang, Y.O. Baker, N. and Kannan, R. (1981) Proc. Natl. Acad. Sci. USA 78, 157-161. 5. Davidson, N.O., Powell, L.M., Wallis, S.C. and Scott, J. (1988) J. Biol. Chem. 263, 13482-13485. 6. Tennyson, G.E., Sabatos, C.A., Higuchi, K., Meglin, N. and Brewer, H.B., Jr. (1989) Proc. Natl. Acad. Sci. USA 86, 500504. 7. Baum, C., Teng, B-B. and Davidson, N.O. (1990) J. Biol. Chem. In press. 8. Davidson, N.O., Carlos, R.C. and Lukascewicz, A.M. (1990) Mol. Endocrinol. 4, 779-785. 9. Wu, J.H., Semenkovich, C.F., Chen, S-H., Li, W-H. and Chan, L. (1990) J. Biol. Chem. 265, 12312-12316. I0. Teng, B-B., Verp, M., Salomon, J. and Davidson, N.O. (1990) J. Biol. Chem. In press. ii. Chapman, M.J. (1986) Methods Enzymol. 128, 70-143. 79
VoI. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12. Black, D.D. and Davidson, N.O. (1989) J. Lipid Res. 30, 207218. 13. Chomczynski, P. and Sacehi, N. (1987) Anal. Biochem. 162, 156159. 14. Driscoll, D.M., Wynne, J.K., Wallis, S.C. and Scott, J. (1989) Cell 58, 519-525. 15. Sanger, F. and Coulson, A.R. (1978) FEBS Lett. 87, 107-110. 16. Knott, T.J., Wallis, S.C., Powell, L.M., Pease, R.J., Lusis, A.J., Blackhart, B., McCarthy, B.J., Mahley, R.W., Levy-Wilson, B. and Scott, J. (1986) Nucleic Acids Res. 14, 7501-7503. 17. Davies, M.S., Wallis, S.C., Driscoll, D.M., Wynne, J.K., Williams, G.W., Powell, L.M. and Scott, J. (1989) J. Biol. Chem. 264, 13395-13398. 18. Chen, S-H., Li, X., Liao, W.S.L., Wu, J.H. and Chan, L. (1990) J. Biol. Chem. 265, 6811-6816.
80
