Apolipoprotein B messenger RNA editing is developmentally regulated in pig small intestine: Nucleotide comparison of apolipoprotein B editing regions in five species

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).

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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.

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