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Liver-Specific RNA Processing of the Ubiquitously Transcribed Rat
Fibrinogen y-Chain Gene
By Patricia J. Haidaris and Mary Anne Courtney
Fibrinogen y-chains differ in amino acid sequence at the
carboxyterminus due t o alternative 3'RNA processing. Previous studies reported differences between humans and rats in
the mechanism of y-chain RNA processing and that it was a
nonregulated event. To test the hypothesis that rat y-chain
RNA processing involves both alternative splicing and poly(A)
site selection and that it is regulated in a tissue-specific
manner, we determined the tissue distribution of y-chain
mRNA expression and the pattern of y-chain pre-mRNA
processing. The results of in situ hybridization demonstrated
that yA and -yB transcripts were localized t o and codistributed in liver hepatocytes, indicating that no subset of cells
process y B mRNA. The ubiquitous expression of the fibrinogen y-chain promoter was demonstrated in marrow, lung,
brain, and liver by RNase protection using a 5'-specific
ychain probe. RNase protection studies t o map 3' RNA
processing sites suggested that, in addition t o the distal
poly(A) signal previously identified, t w o alternative poly(A)
signals within the last intron (ATTAAA and AATAAA) were
used only in liver t o produce yB transcripts. Approximately
equal usage of the three poly(A) signals (27%. 37%. and 36%,
respectively) t o form the 3' end of mature yB transcripts
suggested that poly(A) site selection is random. These results indicate that splicing of the last intron t o produce -yA
mRNA is the ubiquitous and constitutive pattern of ychain
RNA processing, while retention of the last intron t o produce
yB mRNAs is the tissue-specific and regulated pattern of
ychain RNA processing. The pattern of rat y-chain RNA
processing is similar t o human, implying that the mechanism
is conserved. These data support a mechanism of tissuespecific splice site selection predominating over poly(A) site
selection in ychbin pre-mRNA processing. The expression of
both fibrinogen y-chain transcripts in liver, rather than mutually exclusive expression in liver and other tissues, provides a
new model for studying tissue-specific alternative 3' end
formation regulatory mechanisms.
o 1992b y The American Society of Hematology.
F
expression of the quantitatively minor yB-fibrinogen*
polypeptide is hepatocyte-specific in that it is not found in
platelets or their marrow progenitors, megakaryocytes, as is
IBRINOGBN is a dimeric molecule composed of pairs
of three nonidentical subunits, A a and BP and y.' The
fibrinogen chains are encoded by single-copy
Each
gene is characterized by a single transcription initiation
start site; however, each differs at the 3' end of its mRNA.
The A a transcript encodes an additional 15 amino acids at
the carboxyterminus that are not found in the circulating
while a single BP-chain gene product is encoded
by three transcripts differing in length due to multiple
poly(A) site selection.' In contrast, two y-chain transcripts
encode gene products (yA and yB) that differ at the
carboxyterminus due to differential RNA processing.8-'o
Fibrinogen functions that act in support of platelet
aggregation and fibrin clot formation are, in part, mediated
through the y-chain subunit carboxytermini.'.'' The contribution of the alternatively spliced y-chain polypeptide (yB)
in support of platelet aggregation is less effective than the
yA-~hain."-'~We have previously demonstrated that the
From the Depaments of Medicine (Hematology Unit), Microbiology and Immunology, and Pathology and Laboratory Medicine,
University of Rochester School of Medicine and Denhishy, Rochester,
Ny.
Submitted July 23, 1991; accepted October 23, 1991.
Supported in part by Grant No. HL-30616 (P.J.H.)and HL-07152
( M A C . )from the National Heart, Lung and Blood Institute, National
Institutes of Health, Bethesda, MD, and Biomedical Research Support
Grant No. PHS S7RRO5403-25 to Patricia J. (Simpson) Haidark Ffrom
the University of Rochester.
Address reprint requests to Patricia J. Haidark, PhD, Hematology
Unit, Box 610, University of Rochester Medical Center, 601 Elmwood
Ave, Rochester, NY 14642.
The publication costs of this article were defrayed in part by page
charge payment. This adcle must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-49711921 7905-0031$3.00/0
1218
y A-fibrinogen."
The fibrinogen y-chain alternative RNA processing event
is conserved across species8-'o.'6;however, significant differences between the mechanisms of y-chain RNA processing
in rat and human have been reported. In the rat, the
proposed mechanism involves only alternative splicing,
whereas in the human, the mechanism involves a combination of alternative splicing and poly(A) site ~ e l e c t i o n . 8 ~ ' ~ ~ ' ~ ~ ' ~
In addition, since there is concomitant expression of two
differentially processed y-chain transcripts in liver, it has
been suggested that the y-chain alternative RNA processing mechanism is stochastic, or random."
Recently, we demonstrated by Northern blot hybridization that yB mRNA expression is tissue-specific.*' Both yA
and yB transcripts are expressed in the liver, whereas only
yA transcripts are ubiquitously expressed not only in
marrow:o,2' but in nonhepatic and nonhematopoietic tissues as
This is consistent with the ubiquitous expression of the fibrinogen y-chain promoter demonstrated by
From these observations, we proposed that rat
fibrinogen yB RNA processing is not random, but that it is
regulated tissue-specifically in liver by a mechanism similar
to that of human. The data presented in this report suggest
that y-chain pre-mRNA is posttranscriptionally processed
only in the liver into three different lengths of mature yB
transcripts via a mechanism that involves a combination of
alternative splicing and poly(A) site selection equivalent to
that of human. The results support a model for alternative
*The predominant form of y-fibrinogen is termed y, yA =
yMet-412(rat), or y50 = yVal-4ll(human). The alternatively
spliced gene product is termed y', yB = yPro-420(rat), or y57.5 =
yleu-427(human). All expressions of y-chain mRNAs in this
report use the yA and y B terminology.
Blood, Vol79,
No 5 (March I),
1992: pp 1218-1224
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RAT y FIBRINOGEN 3‘ TISSUE-SPECIFIC REGULATION
pre-mRNA processing that involves negative regulation at
the level of splice site commitment.
MATERIALS AND METHODS
Materials. RNA transcription vector, pGEM3Z, bacteriophage
RNA polymerases (Sp6 and T7), and placental RNase inhibitor
(RNasin) were purchased from Promega (Madison, WI), and the
remaining DNA and RNA modifying enzymes were purchased
from Bethesda Research Laboratories (BRL, Gaithersburg, MD).
Tissue culture reagents were purchased from Life Technologies
(Gaithersburg, MD) and chemical reagents from Sigma (St Louis,
MO). Radionuclides were purchased from DuPont New England
Nuclear (Boston, MA).
Constructionof templates and riboprobe synthesis. The rat fibrinogen yA (pyAIB) and yB (pyB) and rat fibronectin (pFN+2)
probes were prepared as previously described?’.’’ The rat fibrinogen y-chain gene 5’ probe (py5‘) was a kind gift from Dr G.
Crabtree, Stanford University.” Riboprobes for RNase protection
assays were prepared at a specific activity of 2.7 x 108 cpm/pg
using [a-’*P]CTP (800 Ci/mmol). For in situ hybridizations, tritiumlabeled probes were prepared at a specific activity of 3.1 x lo7
cpm/pg using both [a-’H]CTP (24.3 Ci/mmol) and [a-’HIUTP
(36.9 Ci/mmol). Antisense transcripts are designated with a “+”
addition to the plasmid name (eg, pyB+ = fibrinogen y B antisense
transcript) and the sense transcripts, which were used as negative
controls, are designated with a “-” addition to the plasmid name
(eg, pyA/B- = fibrinogen yA/B sense transcript).
Preparation of tissue. All animal work was performed according
to protocols approved by the University Committee on Animal
Care and Usage. Rat liver, lung, and brain were removed postmortem and immediately minced, frozen in dry ice, and stored at
-80°C until RNA extraction. Rat tissues extracted for in situ
hybridizations were sliced into 2-3-mm sections and immediately
fixed by submersion in formalin (10% formaldehyde in phosphatebuffered saline).
Cell culture. Faza cells were a kind gift of Dr G. Fuller from the
University of Alabama, Birmingham, and were cultured in DulbecCO’S modified Eagle’s media: Ham’s F12 (1:l) with 5% fetal bovine
serum in 5% COTz6Cells were passaged by trypsinization when
confluent and diluted fivefold in fresh media.
RNA isolation and RNase protection assays. Anticoagulated rat
marrow was collected” and immediately mixed in 5 parts (vol/vol)
RNA lysis buffer and frozen at -80°C to complete lysis of cells?’
Isolation of total and poly(A)’ RNA was p e r f ~ n n e d . ~For
, ~ ’RNase
protection assays, RNA samples were coprecipitated with carrier
yeast tRNA, dissolved in hybridization buffer (40 mmol/L PIPES
(piperzaine-N,N’-bis[2-ethanesulfonic
acid]), pH 6.4, 40 mmol/L
NaCI, 1 mmol/L EDTA, 80% deionized formamide), 1 X lo6 cpm
probe was added, the mix was denatured at 85°C for 5 minutes,
quickly transferred to 45”C, and incubated 12 to 16 hours. Unprotected probe RNA was degraded by RNase A (40 pg/mL) and
RNase T1 (2 Fg/mL) at 30°C for 60 minutesa Protected probe
RNAmRNA heteroduplex was denatured and resolved by electrophoresis in 6% polyacrylamide wedge gels under urea denaturing
condition^.^^ Single-stranded RNA markers were prepared from
pGEM Sp6 and T7 Control Templates (Promega). Calculation of
protected probe lengths using the Sp6 and T7 markers was
reproducible within -t 5 nucleotides (nt). Yeast tRNA was used as
a negative control RNA.
In situ hybridizations. In situ hybridizations were performed as
described previously.” Liver sections were treated with 10 p,g/mL
proteinase K. To determine the relative abundance of mRNAs, the
amount of probe applied was normalized to the shortest probe
sequence as described?’ The relative abundance of yA and yB
1219
transcripts per cell was determined by counting the silver grains
localized to individual hepatocytes from 8 x 10-in color laser
copier enlargements of 2 x 2-in photographic slides of darkfield
images. Only areas representing a cross-section of a whole cell
were counted. Background levels of the pyA/B- and pyB- sense
probes were quantitated in the same manner.”
RESULTS
Distribution and relative abundance of fibrinogen ychain
mRNAs in rat liver. To determine the distribution of cells
expressing the fibrinogen y-chain mRNAs in liver, we used
in situ RNARNA hybridizations. The antisense probes
used for analysis of yA (pyA/B+) and yB (pyB+) transcripts were as described previously.mThe expression of yB
mRNA (Fig IC) was found to parallel the expression of yA
mRNA (Fig 1A). All y-chain transcripts were localized to
the cytoplasm of virtually all hepatocytes (Fig 1C and D
compared with Fig 1A and B). No nuclear accumulation of
incompletely spliced y-chain pre-mRNA was detected using the yB probe, which corresponds to the last intron of
the y-chain pre-mRNA (Fig 1C). These results suggest that
incompletely processed intermediates containing the last
intron do not accumulate in the nucleus in steady-state
levels. In contrast, using this technique and another introncontaining probe, we can demonstrate nuclear localization
of steady-state levels of intron-containing RNA transcripts
(not shown). No other cell type in liver expressed yA or yB
mRNAs such as endothelial (Fig 1, arrowheads) or Kupffer
cells (not shown). Probes were applied to liver sections at
concentrations that normalize for probe complexity (0.3
pg/mL/kb probe complexity) to determine the relative
abundance of the steady-state levels of accumulated transcripts.” To calculate the relative abundance of yB compared with yA mRNA, the silver grain density obtained
with each probe was quantitated and the background levels
subtracted. The background level of silver grain density was
3.6 grains per cell (N = 20) (Fig 1E and F). The relative
abundance of yB mRNA is approximately one third of total
y-chain mRNA (Table l), which is the expected ratio based
on the amount of yB-containing fibrinogen purified from
plasma.w
Ubiquitous expression of the rat y-chain promoter. To
demonstrate the ubiquitous expression of the y-chain
promoter, samples of total RNA isolated from liver, marrow, lung, and brain were hybridized with a 32P-labeled
RNA probe, representing the 5’ end of the rat y-chain gene
(py5’+). The protected fragment (Fig 2, lanes y5’) indicated that the y-chain pre-mRNA was transcribed in each
tissue.
Liver-specific expression of yB transcripts. To determine
which tissues express yB-specific mRNA, the introncontaining probe pyB+ was used. The results demonstrated expression of yB-specific mRNA in liver only. No
yB transcripts were found in marrow, lung, or brain (Fig 2,
lanes yB). To demonstrate the integrity of the mRNA in
each total RNA preparation, we used a fibronectin-specific
probe (pFN+2). An FN-specific protected band was observed using RNA from all tissues tested (Fig 2, lanes FN).
The combined results presented in Fig 2 confirm the
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HAlDARlS AND COURTNEY
1220
Fig 1. In situ hybridizationof liver. Rat liver was formalin-fixedimmediately postmortem,embedded in paraffin, and sectioned. The top panels
are the correspondingdarkfieldview of the lightfieldview shown in the bottom panels. A and B were probed with pyA/B+, C and D were probed
with pS+,and E and F were probed with plA/B-. The bar in F represents 50 pm. A representative hepatocyte cytoplasm (short arrow).
hepatocytenucleus (long arrow), and endothelialcell (arrowhead)are shown in E, D. and F. Antisense probes are designated with a "+" added to
the plasmid name, and the sense probes, which are used as negative controls, are designated with a "-" added to the plasmid name.
tissue-specific expression of yB mRNA in liver only and the
ubiquitous cxprcssion of yA mRNA in liver, marrow, lung,
and brain.
-yB utilizes three difcrent poly@) signab in tissue-specific
alternativeRNA processing To determine the 3' sitcs used
in y-chain RNA processing in liver, we used pyA/B+ and
pyB+ RNA probes (Fig 2A, lanes yA/B and lanes yB,
respectively, and Figs 3 and 4). The interpretation of the
results of RNase protection analysis of y-chain 3' RNA
processing sites using pyA/B+ are shown in Fig 3. The
protected probe fragments of 210/220 nt for exons VIII-IX
and 265 n t for exon X were found, corresponding to the
predicted length of sequences protected by yA transcripts.
The protected probe fragment of 940 nt (Fig 3) corresponds
to the class of y B transcripts representing the YB cDNA
clone,X which include exons VIII-IX, the complete last
intron, and exon X. The poly(A) signal used for this species
ofyB mRNA is also used for YAProcessing and it is bcatcd
in exon x at Position 7340 (see Fig 5). A Putative PolY(A)
signal within the last intron at position 7070 (Fig 5) Was
idcntificd by DNA sequence analysis of the rat y-chain
gene." The protected band of 640 nt suggests that this
Putative POlY(A) signal is used in YB RNA Processing. In
addition, WC O b s m d a Protected band of 560 nt (Fig 31,
Table 1. RelativeAbundance of Steady-State Levels of Fibrinogen
yA and yB Transcripts in Rat Liver
Probe
Silver Grains
per Cell (X)
SE
N
pyA/B+
41.O
2.2
20
PVB+
14.5
1.2
20
'Relative abundance of yA =
tRelative abundance of yB =
[xlD,A~B.l
- x,,,.,/x,,,~,.,I
[xlm.,/x,mA,o.,]
x 100.
Relative
Abundance
64.7%'
35.3%t
x 100.
suggesting that a third sequence is utilized in yB RNA
processing. An analysis of the last intron sequence for
consensus poly(A) sites showed that an additional poly(A)
signal was found at position 6929 ( A T A A A ) (Table 2),
which conforms to the consensus sequence derived from
many species, and to that used in 12% of the known
mRNAs (reviewed by Bernstiel et all'). Polyadenylation at
this position would result in a protected RNA fragment of
557 nt. When the 3' flanking regions for these predicted
poly(A) signals were compared with the consensus sequcnccs," the AATAAA signal is followed within 30 nt by
the dinucleotide CA or CT,and downstream is a GT- or
T-rich motif within another 30 nt as expected (Table 2).
The use of all three poly(A) signals in y B RNA processing
(Fig 5) accounts for all of the protected RNA bands we
observed in the RNAse protection assay (Fig 3), and is
consistent with the relative molecular sizes of the three
polyadenylated yB transcripts, excluding poly(A) tails, that
we identified in liver by Northern hybridization.m In addition, total RNA from the FAZA cell line, a rat hepatocar&
noma, protected the Same population of probe RNA
fragments as did rat liver RNA (Fig 2c).
The pyA/B+ riboprobe contains within its sequences the
Dral fragment (pyB+) specific for yB mRNA. We used
pyB+ to perform RNase protection assays and the results
confimed the interpretation of poly(A) signa[ usage for y~
RNA processing (Fig 4). The 420-nt band corresponds to a
species of RNA that contains all of the intron sequences
rcpresented by the probe. This species of mRNA most
likely represents the yB transcript polyadenylated at position 7340 (Fig 5). The 365-nt band (Fig 4) corresponds to
utilization of the poly(A) signal at position 7070 (Fig5), and
the 270-nt band (Fig 4) corresponds to polyadenylation at
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1221
RAT y FIBRINOGEN 3' TISSUE-SPECIFIC REGULATiON
Fig 2. RNase protection analysis of fibrinogen
?chain 5' and 3' RNA processing sites. Six percent
polyacrylamide-urea denaturing gel electrophoresis
of rat liver (A), marrow (B), brain (D), and lung (E)
total RNAs protecting the following probes: pyA/E+
pvB+ (lanes -@I, and
(lanes yAlE), p+'+(lanes +'),
pFN + 2 (lanes FN) from RNase digestion. All probes
were negative with tRNA, except p+'+,which resulted in a faint protected band in all tissues and is
denoted with the asterisk (A-E, lane +').The long
(+');
arrows point t o the bands protected by *'+
the short arrows point t o bands protected by
and the arrowheads point t o bands protected by
pFN+2 (FN). Additional controls in (C) were protection by plasmid DNA pyAlE with the RNA transcript
pvB+(-@) and FAZA cell total RNA protection of
pyA/E+, FAZA(yA/B). This figure is a composite of
different gels. Molecular sizes of protected bands are
indicated in nucleotides (nt) in the left margin. Markers used for determination of sizes of protected
bands ranged from 42 t o 1,418 n t (not shown). The
faint protected bands observed primarily in (A) lane
yAlE, (C) lane FAZA (yA/E), and (A) lane FN, result
from protection of less than full-length riboprobe
transcripts. The lanes yA/B (A and C) were overexposed t o bring out the 940-nt species. A more representative exposure of liver RNA protecting pyAl B+
can be seen in Fig 3.
A. LIVER
0. MARROW
C. CONTROLS
D. BRAIN
E.LUNG
.$ $@
! t
660 560 e+,
940
465
0 -
*
+
418-
365 -b
265
I
+
-
220
210-
Q
--.)
position 6929 (Fig 5). The relative intensities of the protected bands for pyB+ were determined by densitometric
scanning of autoradiographs within thc linear range of
exposure. The results were corrected for the number of
labeled cytidine residues in each probe fragment (Table 2).
The poly(A) signals at position 6929,7070, and 7340 (Fig 5)
were selected, 37%, 36%, and 27%, respectively, for y B
RNA processing (Table 2).
B
A
A
fulllength +probe
Dra I
Dra I
u
-
W
c
protectedprobe
I
i
t. '
m
a
-420
-265
J=
'210
0
Fig 3. RNase protection analysis of fibrinogen y-chain 3' RNA
processing sites using pyA/B+. (A) Six percent polyacrylamide-urea
denaturing gel electrophoresis of pyA/E+ RNA fragments protected
by rat liver RNAfrom RNase digestion. (E) Full-length pyA/B+ probe.
The Dral fragment is denoted by arrows and sequences of the probe
generated from the multiple cloning region of pGEM3Z denoted by the
wavy lines. (C) The exon (open bars) and intron (line) regions of probe
protected by the mRNA are aligned with the fragment in the gel.
I -270
Fig 4. RNase protection analysis of fibrinogen ychain 3' RNA
processing sites using @+.(A) Polyacrylamide-urea denaturing gel
electrophoresis of pvB+ RNA fragments protected by rat liver RNA
from RNase digestion. (E) Full-length @+ probe. The Dral fragment
is shown and sequences of the probe generated from the multiple
cloning region of pGEM3Z denoted by the wavy lines. (C) The exons
(open bars) and intron (line) regions of probe protected by the mRNA
are aligned with the fragment in the gel.
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HAlDARlS AND COURTNEY
1222
P do,@
IX ' I I X I
Nuclear yChain pre-mRNA
PA PA PA
I
v
II I I I I V
VI1
VI
Vlll
U
Lung
Brain
Nuclear yChain RNA Processing
- II
I
v
Mature, Cytoplasmic yChaln mRNAs
YA
YA
DISCUSSION
In the human, the major y-chain transcript results from
processing of the 10 exons and polyadenylation at the
downstream poly(A) site, while the minor form results from
the inclusion of the last intron, and polyadenylation occurring at a poly(A) site within the intr~n.'~,''
Before this study,
it was thought that the major form (yA) of the rat y-chain
transcript was spliced similarly to the human, but in
processing of the minor form of rat y-chain mRNA (yB),
the last intron was not removed and polyadenylation
occurred at the same downstream poly(A) signal as the yA
transcript: despite the presence of a putative poly(A)
signal in the last intron." However, in this report, we
demonstrate that processing of rat fibrinogen y-chain
pre-mRNA is similar to human, involving both multiple
poly(A) site selection and alternative splicing. Our data
suggest that splicing and polyadenylation of the y-chain
pre-mRNA to produce yA transcripts demonstrates a
ubiquitous and constitutive pattern of RNA processing. In
Table 2. Comparison of Putative Polyadenylation Signals of
Fibrinogen y-Chain t o Consensus Polyadenylation Sequence in yB
RNA Processing
Consensus Polyadenylation Signal'
AATAAA...(10-20 nt)...CA......TGT (30nt)
...
ATTAAA (29)
...CA......GT
% Relative Usage f SE
(N = 4)
37.4t 0.1
I
nt 6929t
AATAAA.. .(21)..EA ......GT
35.6 2 2.5
I
nt 7070
AATAAA...( 12)...CT...
27.0 2 2.5
I
nt 7340
*Consensus polyadenylation signal taken from Birnstiel et al?'
tNucleotide (nt) position of ?-chain pre-mRNA.
Fig 5. Proposed mechanism for the posttranscriptional regulation of fibrinogen y-chain 3' pre-mRNA
processing. The y-chain pre-mRNA is shown drawn t o
scale. Wide, open boxes depict exon sequences, and
lines depict intron sequences. The last exon is depicted as a closed box, and the last intron, which
becomes an exon, is depicted as a narrow open box.
The poly(A) signals (PA), which were mapped by
RNase protection analysis, are shown at the nucleotide positions of 6929, 7070. and 7340 of the premRNA. The nuclear expression of the ubiquitous
y-chain pre-mRNA is depicted at the top. The constitutive pattern of y-chain pre-mRNA processing is
shown in marrow, lung, and brain (right-center). Only
mature yA mRNA is produced (right-bottom), which
is localized in the cytoplasm of select cell types. The
tissue-specific regulated pattern of y-chain premRNA processing is shown in liver (left-center). The
yA and three yB transcripts of different lengths (leftbottom) are found in the cytoplasm of liver hepatocytes. The putative liver-specific splicing repressor
(stippled oval) would be in competition with the
ubiquitous cellular splicing machinery (striped oval)
during y-chain pre-RNA processing.
contrast, retention of the last intron and polyadenylation of
the y-chain pre-mRNA to produce yB transcripts demonstrates a tissue-specific pattern of RNA processing.
The key regulatory event controlling the abundance of
mRNAs with differentially processed 3' ends can occur at
one or more steps: transcription termination, differential
RNA stability, poly(A) site selection, or splice site commitment. The formation of 3' ends of eukaryotic mRNAs are
usually generated by RNA processing and not transcription
termination per ~ e . ~ ' -Since
"
a distinct population of yB
transcripts are processed at the same poly(A) signal in exon
X as yA transcripts, it seems unlikely that a difference in
stability of the processed transcripts is the regulatory
mechanism. Some models of regulation of alternatively
3'-end processed genes proposed that poly(A) site selection
predominated over splice site ch~ice.'~,'~
In the case of the
fibrinogen y-chain transcription unit, if poly(A) site selection were the key regulatory step, then the mechanism
would have to distinguish between utilization of the distal
poly(A) site in exon X for yA versus y B processing. Once
the poly(A) site is selected, a splice or not-to-splice choice
must be made as well. It is difficult to reconcile a poly(A)
site selection regulatory mechanism that would accommodate these choices.
Recent models of regulation of alternatively 3'-end
processed genes propose that splicing or splice site commitment can occur before 3' endonucleolytic cleavage and
p~lyadenylation.~~-~'
The simplest explanation of our data is
that splice site commitment, not poly(A) site selection, is a
key regulatory step in the liver-specific processing of the
y-chain pre-mRNA. The observations from this study
suggest that fibrinogen y-chain gene expression is regulated, not stochastic. We propose that the regulatory
mechanism involves a liver-specific factor that acts as a
repressor to regulate the splicing of the last intron, as
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1223
RAT y FIBRINOGEN3’ TISSUE-SPECIFIC REGULATION
shown in Fig 5 . In the liver, the splicing repressor (stippled
oval) would be in competition with the splicing commitment
machinery (striped oval). When splicing of the last intron is
inhibited by the putative, trans-acting splicing repressor,
the production of mature yB transcripts would follow.
Polyadenylation would occur at any of the three different
poly(A) signals. The choice of polyadenylation signal used
would have no effect on the final translational reading
frame of mature yB mRNA, and thus would be silent in the
overall expression of the yB polypeptide chain in plasma
fibrinogen. This mechanism proposes a negative control of
y-chain pre-mRNA splicing, which is consistent with the
known mechanisms of alternative pre-mRNA splicing reviewed by mania ti^.^' To date, there is no example in which
alternative splice site selection is under positive control.4l
The precedent for splice site over poly(A) site selection as a
means of developmental or tissue-specific regulation has
been suggested for two well-studied alternatively processed
complex transcription units: the rat calcitonin/calcitonin
gene-related peptide (CGRP) and adenovirus transcription
Cis-acting elements near the calcitonin-specific-3’splice junction have been shown to regulate the alternative
splicing of the calcitonin/CGRP gene, and it has been
proposed that these sequences serve to inhibit the production of calcitonin transcripts in CGRP-producing cells.4’
Trans-acting nuclear factors have been implicated in the
regulation of these developmental or tissue-specific RNA
processing s t e p ~ . ~ ~ ’We
~ ~are
’ ~ ‘currently
~”
investigating the
cis- and trans-acting factors involved in fibrinogen y-chain
pre-mRNA processing using eukaryotic transfection systems in heterologous, hepatoma and megakaryocyte cell
lines.
The ubiquitous expression of the y-chain gene demonstrated by uszoand others22-24
suggests that the expression of
the fibrinogen y-chain transcript compared with the Aa-
and BP-chain transcripts is noncoordinated in nonhepatic
tissues. Therefore, the results presented in this study are
not incompatible with the recent evidence by others demonstrating uptake4’,* and not synthesis of fibrinogen in megakaryocyte~.4’-~~
The functional significance of the ubiquitous
expression of the y-chain of fibrinogen is currently under
investigation.
The fibrinogen y-chain gene has not been recognized as a
model for the study of tissue-specific regulation by 3‘ RNA
processing events. Tissue-specificand developmental expression of eukaryotic genes is frequently regulated by alternative RNA pro~essing.”~~~
Typically, genes regulated by
alternative 3’ processing are mutually exclusively expressed
in tissues or cell types, or developmentally expressed. The
expression of both fibrinogen y-chain transcripts in liver
hepatocytes, rather than mutually exclusive expression in
liver and other tissues, provides a new model for studying
alternative 3’ RNA processing regulatory mechanisms. The
y-chain model is unique in that only one gene of the three
genes of a complex, multimeric protein is subjected to 3’
tissue-specific regulation. The only khown functions of
yB-fibrinogen which differ from yA-fibrinogen are a reduced ability to support adenosine diphosphate-induced
platelet aggregati~n’”’~
and an inability to cause staphylococcal clumping.’’ The change in function and the evolutionarily conserved compartmentalization of yB-fibrinogen to
plasma suggests a need for clotting associated with reduced
platelet aggregation or adhesion.
ACKNOWLEDGMENT
The authors thank Laurie Neroni, Melissa Vanek, and Barbara
Earnest for technical assistance, Kristin Leibert for literature work,
and Carol Weed for help in preparation of the manuscript. The
authors also wish to acknowledge Dr Stuart Horowitz for critical
reading of the manuscript.
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1992 79: 1218-1224
Liver-specific RNA processing of the ubiquitously transcribed rat
fibrinogen gamma-chain gene
PJ Haidaris and MA Courtney
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