Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 1489-1493, February 1991
Genetics
In vitro apolipoprotein B mRNA editing: Identification of a 27S
editing complex
(cytidine/uridine "*editosome"/ mooring sequence)
HAROLD C. SMITH*†‡$, SHU-RU Kuot, JOHN W. BACKUst, STANLEY G. HARRIS*‡, CHARLES E. SPARKS*
AND JANET D. SPARKS*
Departments of *Pathology and Laboratory Medicine. *Biochemistry, and ‡The Cancer Center, University of Rochester, 601 Elmwood Avenuc, Rochester.
NY 14642
Communicated by Fred Sherman, November 14, 1990 (received for review June 14, 1990)
ABSTRACT
Specific apolipoprotein B (apoB) mRNA ed-
iting can be performed in vitro on apoB RNA substrates. Native
gels and glycerol gradient sedimentation have been used to
determine the physical properties of the in vitro editing activity
in rat liver cytosolic S100 extracts. ApoB RNA substrates were
progressively assembled as 27S complexes for 3 hr with similar
kinetics as seen for the accumulation of edited RNA. Assembly
was not observed on RNAs from apoB deletion constructs that
did not support editing. The 27S complex contained both edited
and unedited RNA sequences. Inhibition of 27S complex as-
sembly by vanady|-ribonucleoside complexes was accompa-
nied by inhibition of editing. Based on these data, we propose
that the 27S complex is the in vitro "editosome." A "mooring
sequence" model for RNA recognition and editosome assembly
has been proposed involving RNA sequences flanking the edited
nucleotide.
Apolipoprotein B (apoB) is translated from a 14-kilobase (kb)
mRNA that is transcribed from a single-copy gene on human
chromosome 2 (1, 2). In rats (3-6) and humans (7), apoB
exists as a high molecular weight form (apoBH or apoB100)
and a low molecular weight form (apoB or apoB48). Al-
though apoB is synthesized from the same primary tran-
script as apoBH, its mRNA undergoes an unusual form of
RNA processing referred to here as "C/U RNA editing
wherein a cytidine at nucleotide 6666 is converted to a uridine
(8-11). This transition alters the sense of codon 2153 from
glutamine (CAA) to a translation stop (UAA). The contribu-
tion of apoBH very low density lipoprotein (VLDL) to low
density lipoprotein (LDL; an atherogenic risk factor) through
metabolic conversion in the plasma has focused attention on
the various metabolic levels where the apoBH/apoB ratio
correspondingly reduced to below detectable levels, while
apoB, synthesis remains relatively unaltered.
To a first approximation, apoB mRNA editing appears
similar to plant mitochondrial editing (20-22), which primar-
ily involves cytidine-to-uridine conversions under conditions
where the reading frame is maintained. It is believed that C/U
editing might be catalyzed by a form of cytidine deaminase
(23, 24), although other possibilities have not been ruled out.
Hemoflagellate mitochondrial mRNA editing differs from
apoB mRNA editing in that it involves insertion and deletion
of single or multiple uridine residues at single and multiple
sites (25-27). This form of editing results in extensive mod-
ification of mRNAs and their reading frames. Paramyxovirus
mRNA editing involves a different mechanism, wherein
single guanidine nucleotides are inserted at select sites within
a subset of mRNAs to produce major changes in the reading
frame (28).
Point mutant constructs of the apoB editing site suggest a
lax sequence requirement within the immediate vicinity of the
edited nucleotide (23). Mutants which place additional cy-
tidines adjacent to the editing site were edited at these
additional cytidine residues. The paradox of the editing
activity having absolute specificity for a single nucleotide yet
lacking selectivity when confronted with multiple cytidine
residues within the region suggests that editing specificity is
achieved by the positioning of the editing activity over the
correct nucleotide. A role for more distal sequences in this
process is suggested by deletion-mutant studies (24, 29, 30).
ApoB mRNA deletion constructs ranging in size from 2.4 kb
to 26 base pairs (p) were both expressed and edited in
transient expression assays with McArdle 7777 cells (29). In
contrast, transcripts from these subclones are all edited in
vitro bv McArdle cell cvtosolic S100 extracts excent the
Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 1489-1493, February 1991
Genetics
In vitro apolipoprotein B mRNA editing: Identification of a 27S
editing complex
(cytidine/uridine "*editosome"/ mooring sequence)
HAROLD C. SMITH*†‡$, SHU-RU Kuot, JOHN W. BACKUst, STANLEY G. HARRIS*‡, CHARLES E. SPARKS*
AND JANET D. SPARKS*
Departments of *Pathology and Laboratory Medicine. *Biochemistry, and ‡The Cancer Center, University of Rochester, 601 Elmwood Avenuc, Rochester.
NY 14642
Communicated by Fred Sherman, November 14, 1990 (received for review June 14, 1990)
ABSTRACT
Specific apolipoprotein B (apoB) mRNA ed-
iting can be performed in vitro on apoB RNA substrates. Native
gels and glycerol gradient sedimentation have been used to
determine the physical properties of the in vitro editing activity
in rat liver cytosolic S100 extracts. ApoB RNA substrates were
progressively assembled as 27S complexes for 3 hr with similar
kinetics as seen for the accumulation of edited RNA. Assembly
was not observed on RNAs from apoB deletion constructs that
did not support editing. The 27S complex contained both edited
and unedited RNA sequences. Inhibition of 27S complex as-
sembly by vanady|-ribonucleoside complexes was accompa-
nied by inhibition of editing. Based on these data, we propose
that the 27S complex is the in vitro "editosome." A "mooring
sequence" model for RNA recognition and editosome assembly
has been proposed involving RNA sequences flanking the edited
nucleotide.
Apolipoprotein B (apoB) is translated from a 14-kilobase (kb)
mRNA that is transcribed from a single-copy gene on human
chromosome 2 (1, 2). In rats (3-6) and humans (7), apoB
exists as a high molecular weight form (apoBH or apoB100)
and a low molecular weight form (apoB or apoB48). Al-
though apoB is synthesized from the same primary tran-
script as apoBH, its mRNA undergoes an unusual form of
RNA processing referred to here as "C/U RNA editing
wherein a cytidine at nucleotide 6666 is converted to a uridine
(8-11). This transition alters the sense of codon 2153 from
glutamine (CAA) to a translation stop (UAA). The contribu-
tion of apoBH very low density lipoprotein (VLDL) to low
density lipoprotein (LDL; an atherogenic risk factor) through
metabolic conversion in the plasma has focused attention on
the various metabolic levels where the apoBH/apoB ratio
correspondingly reduced to below detectable levels, while
apoB, synthesis remains relatively unaltered.
To a first approximation, apoB mRNA editing appears
similar to plant mitochondrial editing (20-22), which primar-
ily involves cytidine-to-uridine conversions under conditions
where the reading frame is maintained. It is believed that C/U
editing might be catalyzed by a form of cytidine deaminase
(23, 24), although other possibilities have not been ruled out.
Hemoflagellate mitochondrial mRNA editing differs from
apoB mRNA editing in that it involves insertion and deletion
of single or multiple uridine residues at single and multiple
sites (25-27). This form of editing results in extensive mod-
ification of mRNAs and their reading frames. Paramyxovirus
mRNA editing involves a different mechanism, wherein
single guanidine nucleotides are inserted at select sites within
a subset of mRNAs to produce major changes in the reading
frame (28).
Point mutant constructs of the apoB editing site suggest a
lax sequence requirement within the immediate vicinity of the
edited nucleotide (23). Mutants which place additional cy-
tidines adjacent to the editing site were edited at these
additional cytidine residues. The paradox of the editing
activity having absolute specificity for a single nucleotide yet
lacking selectivity when confronted with multiple cytidine
residues within the region suggests that editing specificity is
achieved by the positioning of the editing activity over the
correct nucleotide. A role for more distal sequences in this
process is suggested by deletion-mutant studies (24, 29, 30).
ApoB mRNA deletion constructs ranging in size from 2.4 kb
to 26 base pairs (p) were both expressed and edited in
transient expression assays with McArdle 7777 cells (29). In
contrast, transcripts from these subclones are all edited in
vitro bv McArdle cell cvtosolic S100 extracts excent the