Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3952-3961
The Ig Heavy Chain 3
End Confers a Posttranscriptional
Processing Advantage to Bcl-2-IgH Fusion RNA in t(14;18) Lymphoma
By
Alexander Scheidel Petrovic,
Robert L.Young, Bernadette Hilgarth,
Peter Ambros,
Stanley J. Korsmeyer, and
Ulrich Jaeger
From the Department of Medicine I, Division of Hematology, University
of Vienna Medical School, Vienna, Austria; the Howard Hughes Medical
Institute, Washington University School of Medicine, St Louis, MO; and
CCRI, St Anna Children's Hospital, Vienna, Austria.
 |
ABSTRACT |
The chromosomal translocation t(14;18) in lymphoma leads to an
overproduction of the Bcl-2 protein on the basis of increased Bcl-2
mRNA levels. Whereas the juxtaposition of Bcl-2 with the Ig heavy chain
locus causes a transcriptional activation, 70% of the lymphomas also
produce Bcl-2-Ig fusion RNAs with Ig 3
ends. Using S1 nuclease
protection assays that can discriminate between nuclear RNA precursors
and spliced mRNA, we found that the fusion RNAs in t(14;18) cell lines
exhibit an additional posttranscriptional processing advantage.
Transfection experiments with artificial genes containing various Bcl-2
or Ig 3 ends show that this effect is (1) related to RNA
splicing and/or nucleocytoplasmic transport; (2) independent of
transcriptional activation by the heavy chain enhancer; (3) dependent
on the presence of the JH-CH and C-
1 Ig
introns; and (4) tissue specific for B cells. This constitutes a novel
mechanism of oncogene deregulation unrelated to transcriptional activation or half-life prolongation. The data further support the
existence of a tissue-specific posttranscriptional pathway of Ig
regulation in B cells.
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INTRODUCTION |
THE CHROMOSOMAL translocation
t(14;18)(q32;q21) constitutes the molecular hallmark of human
follicular lymphoma. The breakpoint on the derivative 14 chromosome
juxtaposes the Bcl-2 proto-oncogene from 18q21 with one of six Ig heavy
chain joining (JH) regions of 14q32.1-3 The
pathologic consequence is the overexpression of both Bcl-2 RNA and
protein in cells bearing this translocation.4-6 Transgenic
mice bearing a Bcl-2-Ig minigene established that this translocation
was of primary pathogenic importance in lymphomagenesis. Bcl-2 mice
demonstrate B-cell follicular hyperplasia that progresses to high-grade
lymphoma.7,8
The breakpoints on chromosome segment 18q21 are not randomly
distributed. Approximately 70% occur within the major breakpoint region (mbr), where most cluster within 150 nt.1-3 Up to
20% of breakpoints are found approximately 30 kb further telomeric within the minor breakpoint region (mcr).9,10 Although both breakpoints are associated with follicular lymphoma, important questions remained concerning the precise mechanisms that deregulate Bcl-2 production. We and others have shown that the rate of Bcl-2 transcription is increased in t(14;18)-bearing cell lines and constitutes one mechanism of deregulation.6,11,12 However, whether the magnitude of this transcriptional enhancement fully accounts for the log-fold increase in steady-state levels of Bcl-2-Ig fusion RNA within t(14;18) cells is uncertain.
Many interchromosomal translocations responsible for neoplasia generate
fusion RNAs between genes on their chromosomal partners. Notable
examples include BCR-ABL,13 PML-RAR
,14
DEK-CAN,15 LYT-10-C1,16 and
MLL-AF4,17 to name a few. Similarly, breakpoints within the
mbr but not the mcr of Bcl-2 result in a Bcl-2-Ig fusion RNA.4,6,9,10 However, one notable difference exists between the Bcl-2-Ig example and most of the aforementioned fusion RNAs. Most
fusion RNAs found in neoplasias encode a chimeric protein product
providing a clear rationale for the selection of clones bearing these
events. In contrast, the mbr of Bcl-2 is located within the 3
untranslated region of the gene and translocations into it result in
Bcl-2-Ig hybrid RNA but not a chimeric protein. Yet, the majority of
Bcl-2 translocations involve this region and generate Bcl-2-Ig fusion
RNAs, suggesting that this event may also have a selective advantage.
The most obvious potential mechanism would be an alteration in mRNA
half-life. This was attractive, because the native Bcl-2 RNA had a
relatively short half-life of 2.5 hours within B cells,6
whereas Ig RNAs demonstrated a longer half-life approaching 20 hours.18,19 However, experiments showed that the Bcl-2-Ig
fusion RNA exhibited an unaltered half-life of 2.5 hours.6
Posttranscriptional control by differential RNA splicing or transport
has been documented for a variety of genes. A number of organisms show
tissue specific splicing.20-22 Cellular transport mechanisms have been proven to be important in the expression of
viruses such as human T-lymphotrophic virus (HTLV) I and
II,23-25 influenza virus NS1,26 or hepatitis
B.27,28 The existence of a posttranscriptional pathway
related to intronic sequences has also been postulated for Ig RNA
regulation.29 These observations prompted us to examine the
processing of Bcl-2-Ig fusion versus native Bcl-2 RNAs. We developed a
system that eliminated the potential influence of transcriptional
initiation to examine the contribution of Ig versus Bcl-2 sequences on
posttranscriptional control. This assay, which discriminates between
precursor and spliced mRNA products, defined a processing advantage for
Bcl-2-Ig fusion RNA, a novel mechanism of oncogene activation. Because
the improvement is conferred by Ig sequences and is tissue specific for
B cells, it may also contribute to the efficiency of normal Ig gene
expression.
| |
MATERIALS AND METHODS |
Cell culture.
Cell lines were maintained in Iscove's modified Dulbecco's medium
(GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum, penicillin, streptomycin, and 50 mmol/L 
-mercaptoethanol at a
CO2 concentration of 7.5%. The following cell lines were used: Nall-1 (pre-B30); SU-DHL9 (mature B); SU-DHL-6
[t(14;18) bearing mature B31]; K562 (erythroleukemia);
70z/3 (mouse pre-B32); and S194 (mouse myeloma, ATCC TIB
10, 1985).
Construction of plasmids and transfection.
A 1.4-kb Sst I/BamHI human -actin promoter fragment
was excised from pHAPr-I (kindly provided by L. Kedes33,34). This fragment contains 472 nt of 5
flanking region, the -actin promoter, exon I, IVS I including the
-actin enhancer, and an acceptor splice site and has been shown to
retain 78% of promoter activity when transfected into Hela
cells.35
To generate the backbone of the Apr-fusion vectors, this fragment
was supplied with an Sst I linker on both ends and inserted into the Sst I site of Bluescript (Stratagene, La
Jolla, CA) pKS (T7 = 5; termed plasmid p229.6; see Fig 2). Thus,
95 nt of polylinker from the acceptor splice site to the unique
EcoRI site become part of exon II of this vector. The different
3 ends include (1) a 5.6-kb EcoRI genomic fragment from
a normal Bcl-2 allele 3 of the EcoRI site (at position
1856)3 in Bcl-2 exon III1,2,6
(Apr-Bcl-2); (2) an 11.6-kb EcoRI genomic fragment
from a Bcl-2-Ig fusion allele (SU-DHL-62;
Apr-Ig); (3) a 2.4-kb EcoRI cDNA fragment from
SU-DHL66 fused to the C-1 membrane genomic 3 end
including its polyadenylation signal (Apr-Ig
Introns); and
(4) a 2.5-kb EcoRI cDNA fragment as in (3) with the exception
that a 134 nt non-Ig intron (IVS II of the human -actin gene) was
inserted into the 2.4-kb EcoRI cDNA as a substitute for the
JH6 to C-1 intron (Apr-Ig
Intron). These
fragments were inserted into the EcoRI site of p229.6 to form
the final Apr-fusion vectors. All constructs were partially sequenced to identify the correct orientation and checked for single-copy integration by restriction mapping.
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| Fig 2.
Constructs for transfection. A 1.4-kb Sst I (S)
fragment of the human -actin promoter plus -actin IVS-I was fused
to various 3 ends at the EcoRI (R) site:
APr-Bcl-2 contains the normal genomic Bcl-2 3 end,
including the Bcl-2 poly(A) sites and 3 flanking regions;
APr-Ig contains a translocated allele cloned from SU-DHL-6,
including the Bcl-2 mbr as well as the JH-CH
and CH introns and the C1 poly(A) sites and flanking
regions; APr-Ig Introns contains a translocated cDNA from
SU-DHL-6 and the genomic C1 membrane (M) poly(A) signal and flanking
regions; Apr-IgIntron corresponds to
APr-IgIntrons with the exception that a nonlymphoid intron (IVS-II of -actin) is inserted as a substitute for the JH-CH intron. The transcriptional start site is
indicated by an arrow. The EcoRI (R) and Xba I (X)
sites were used for the S1 protections and primer extensions on the
-actin promoter.
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Cell transfection and selection of stable integrants.
Transfection of human and murine cell lines was performed by
electroporation with a 1.9-mm gap cuvette electrode and a Transfector 300 (Biotechnologies & Experimental Research, Inc, San
Diego, CA). Log phase cells (1 × 107) were washed and
resuspended in 400 µL of serum-free RPMI 1640 (GIBCO). They were
mixed with 100 µL RPMI 1640 containing 5 µg of RSVneo36
and a 2 molar excess of linearized construct DNA together with 125 µg
of salmon sperm DNA. Transfection of the various cell lines was
performed at the optimal voltage and capacitance (200 to 250 V and 450 to 800 µF). After 24 to 48 hours, cells were selected in 1 g/L of
G418 (Geneticin; GIBCO). Whereas half of each transfection was selected
as a bulk, the other half was plated in 2-mL tissue culture wells at a
density of 103 to 104 cells to obtain
oligoclonal subpopulations. Transfectants were harvested after 10 to 14 days and assayed for construct expression by S1 nuclease protection.
RNA preparation.
For the preparation of nuclear and cytoplasmic RNA, cells were lysed in
a hypotonic buffer containing 0.5% NP-40 and separated on a sucrose
gradient.37 Cytoplasmic RNA from the supernatant was
prepared by this standard protocol, including DNaseI digestion. The
nuclear pellet was resuspended in 4 mol/L guanidine thiocyanate and
nuclear RNA was purified on a 7.5 mol/L CsCl2
gradient.38 Total RNA from transfected cells for run-on
assays was prepared by a guanidine-thiocyanate-acid-phenol miniprep
method.39 S1 probes that can discriminate between RNA and
DNA detected no DNA contamination in these minipreps.
DNA probes for S1 nuclease protection.
A 406-nt HindIII/HincII fragment across the Bcl-2
intron II/exon III acceptor splice site and a 410-nt Sst
I/Bgl II fragment across the Bcl-2 exon II/intron II donor
splice site were subcloned into m13 to generate single-stranded,
synthetically labeled probes. A 183-nt BamHI/Sal I
human -actin cDNA fragment40 was protected as a control
for RNA amount within the same tube.
The probes for the protection assay on RNA from transfected cell lines
were derived from p229.6. This plasmid was end labeled at the
EcoRI or Xba I site and used to detect the correct
-actin initiation site. A 145-nt Bal I/EcoRI
fragment spanning the acceptor splice site in the polylinker was
subcloned into Bluescript pKS and end labeled at the EcoRI
site, giving a probe length of 2.9 kb, a precursor protection of 145 nt, and an exon protection of 95 nt.
S1 nuclease protection assay.
S1 protection with single-stranded reverse complementary DNA probes was
performed as described.6 5 end labeled probes were
generated using 1 µg of linearized and dephosphorylated plasmid, 60 U
of T4-polynucleotide kinase (US Biochemicals, Cleveland, OH), and 100 µCi of [-32 P] ATP
(Amersham, Arlington Heights, IL).41 RNAs were
hybridized overnight with 2 × 105 cpm at
the appropriate temperature (52°C) in 15 µL of a juice containing
80% formamide, 40 mmol/L PIPES (pH 6.4), 400 mmol/L NaCl, and 1 mmol/L
EDTA. Samples were digested with 200 U S1 nuclease (Boehringer
Mannheim, Indianapolis, IN) and analyzed on 6%
polyacrylamide gels.
Nuclear run-on and primer extension assays.
Log phase cells (5 × 107) were washed in RPMI 1640 and resuspended in 10 mL ice-cold hypotonic lysis buffer (10 mmol/L
HEPES, pH 8.0, 1.5 mmol/L MgCl2, 10 mmol/L KCl). After 10 minutes on ice, cells were lysed by two to three passages through a 22G
needle. Nuclei were washed and resuspended in 220 µL of transcription buffer containing 20 mmol/L Tris (pH 8.0), 6 mmol/L Mg
(C2H3O2)2, 84 mmol/L
KCl, 10 mmol/L NH4Cl, 0.3 mol/L EDTA, and 10% glycerol. The run-on assay was performed as described6 at 30°C
for 30 minutes using 250 µCi of [-32 P] GTP as
label. After the reaction was completed, RNA was extracted, ethanol-precipitated, and resuspended in hybridization buffer (50%
formamide, 4× SSC, 2× Denhardt's solution, 20 µg/mL tRNA [Escherichia coli], 50 mmol/L NaPO4 [pH 7.4],
and 0.1% sodium dodecyl sulfate [SDS]). Equal counts (2 × 106 cpm) were hybridized to 2 µg of slot-blotted single
(histone H4) or double-stranded (-actin, RSVneo) DNA template for 36 hours at 42°C. Membranes were washed three times for 20 minutes at
room temperature with 2× SSC, 0.1% SDS and twice for 20 minutes
at 63°C with 0.1% SSC, 0.1% SDS.
Primer extensions were performed as described6 using RNAs
from transfected cell lines as template and a 20-nt oligonucleotide primer starting from the EcoRI site in the polylinker of
p229.6.
RNA half-life experiments.
Actinomycin D (Sigma, St Louis, MO) was added to cell lines growing in
log-phase at a concentration of 10 µg/mL. RNA was extracted after 0, 1, 2, 4, 8, and 24 hours and analyzed by S1-nuclease protection.
| |
RESULTS |
Altered posttranscriptional processing of Bcl-2-Ig fusion RNAs in
t(14;18) cell lines.
S1 nuclease protection assays that cross the Bcl-2 exon II-intron II or
intron II-exon III borders were developed to determine the relationship
between the levels of Bcl-2 nuclear precursors, nuclear spliced
message, and final cytoplasmic mRNA in mature B-cell lines with or
without the translocation (Fig 1A and B). This assay uses probes across the intron-exon borders, thereby hybridizing to the long, unspliced precursor as 406 nt (Fig 1A) or 410 nt (Fig 1B) fragments as well as the shorter spliced mRNA as 140-nt
(Fig 1A) or 180-nt (Fig 1B) fragments. Precursors should only be
visible in the nuclear lanes, whereas the spliced message is detected
in the nucleus as well as cytoplasm. Bcl-2 transcription has previously
been shown to be increased in the t(14;18)-bearing cell line SU-DHL-6,
when compared with the nontranslocated SU-DHL-9 line.6 As
shown in Fig 1A and B, the steady-state level of nuclear precursors was
only slightly greater in SU-DHL-6 compared with SU-DHL-9 as detected by
S1 nuclease protection. However, the amount of spliced cytoplasmic
message was fivefold to 10-fold higher in SU-DHL-6 compared with
SU-DHL-9. This occurred even though the Bcl-2-Ig RNA half-life
remained unchanged after translocation.6 An approximately
threefold increase in spliced nuclear message was noted in SU-DHL-6
versus SU-DHL-9. In addition, a twofold to threefold increase in the
ratio of cytoplasmic to nuclear spliced RNA was noted in SU-DHL-6
compared with SU-DHL-9. These values were measured densitometrically
and corrected to an equalized -actin signal. Using a separate S1
nuclease protection assay for -actin, which detected -actin
precursors, we verified that both cell lines processed -actin
pre-mRNA in an equivalent fashion (data not shown). These observations
suggest a distinct posttranscriptional advantage for the Bcl-2-Ig
fusion transcripts compared with the normal Bcl-2 transcripts at the
level of splicing and/or nucleocytoplasmic transport.
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| Fig 1.
Comparison of nuclear precursors, spliced nuclear
mRNA, and cytoplasmic RNA. Single-stranded S1 nuclease protection
probes and protected fragments are shown at the bottom. The m13-probes were uniformly labeled. The lack of visible bands at nt 735 (Bcl-2) and
nt 183 (-actin) indicates that nuclease digestion is complete and
that only RNA is detected. Cohybridization of the same sample with a
-actin probe controls for equal amounts of RNA. Note that the
-actin probe used here contains only exon sequences (no precursor). (A) Bcl-2 intron II-exon III border. (B) Exon II-intron II border. Precursor protection of the upstream exon is much stronger and probably
reflects increased polymerase loading at the 5 end.
|
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Processing constructs identify a posttranscriptional RNA processing
advantage for an Ig versus Bcl-2 3 end.
A series of constructs was designed to test the influence of various
Bcl-2 or Bcl-2-Ig molecules on posttranscriptional processing (Fig 2). To eliminate any differential
influence from Bcl-2 promoter activity, all constructs used an
identical strong promoter/enhancer system, human
-actin.34 Because -actin, Bcl-2, and Ig genes are
well conserved and function cross-species,7,33 we generated human based processing constructs and transfected them into murine target cells. All constructs contain an intron (IVS I of -actin) to
insure that each transcript is targeted to a spliceosome for processing. APr-Bcl-2 or APr-Ig constructs were
cotransfected with RSV Neo R vector into the S194 murine plasmacytoma
cell line by electroporation. Stable transfectants were selected with
G418 and demonstrated comparable rates of transcription from either integrated construct when assessed by nuclear run-on analysis (Fig 3). The presence of the Ig heavy chain
enhancer did not further augment newly initiated transcription in this
system.
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| Fig 3.
Run-on assays showing equal transcriptional activity of
APr-Bcl-2 and APr-Ig constructs in stable
transfectants of a mouse B-cell line. Nascent, labeled RNA extracted
from S194 cell lines transfected with APr-Bcl-2, APr-Ig,
or only the RSVneo vector was hybridized to DNA probes
detecting the human -actin RNA produced by the Bcl-2 and Ig
constructs (upper row), the RSVneo RNA produced by all three cell lines
transfected with the neomycine resistance gene (middle row), and the
mouse histone H4 gene integral to the S194 cell line (lower row). The
145-nt -actin fragment (p240.1) is specific for the
APr-constructs and is therefore only seen in lanes 1 and 2.
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|
An S1 protection assay designed to assess the 5 region of human
-actin showed that both the APr-Bcl-2 and
APr-Ig constructs demonstrated correctly initiated
transcription (Fig 4A). A few minor
initiation sites were noted surrounding the -actin promoter in the
-APr-Ig cell lines (Fig 4A). Two additional minor start sites were
mapped by primer extension analysis to IVS I, immediately upstream of
the acceptor splice site (Fig 4B). S1 analysis indicated that these
intron-initiated sites (denoted as 117 and 125) were slightly augmented
by the Ig enhancer (Fig 5). However, the
vast majority of transcripts were still correctly initiated upstream.
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| Fig 4.
Mapping construct RNA initiation sites. (A) S1
protections showing comparable amounts of transcripts correctly
initiated at the human -actin promoter start site (nt +1
corresponding to a protection of 975 nt). A few minor start sites
upstream of the promoter and in the IVS-I are flushed on in the
APr-Ig cell line. The 1.4-kb human -actin promoter
fragment (34) in bluescript (p229.6, see the Materials and Methods) was
end-labeled at the EcoRI site and used for S1-protection of the
transfected human allele. (B) The primer extension assay verifies the
location of additional intron start sites in the APr-Ig cell
lines, which are also seen as the middle bands on the S1 protection in
Fig 5 (APr-Ig). A 20-nt primer starting from the
EcoRI site of the -APr plasmid (p229.6) was used.
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| Fig 5.
Lineage specificity and intron dependency. The fragments
protected by the end-labeled S1 probe are shown at the bottom. Total RNA from transfected cell lines was used. (A) Mature B-cell line; (B)
pre-B-cell line; (C) non-B-cell (myeloid) line; (D) mature B-cell
transfected with the APr-IgIntrons and
APr-IgIntron constructs.
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Stable integrants of the APr-Bcl-2 or APr-Ig
constructs within the S194 plasmacytoma cell line were assessed for
their efficiency of RNA processing. The inclusion of the -actin
intron within these constructs enabled the creation of a generic S1
protection assay that could distinguish nuclear precursors from spliced
RNA (Fig 5). Both populations of stably transfected S194 cells showed comparable amounts of nuclear precursor RNA from either
APr-Ig or APr-Bcl-2 constructs. Yet, the
constructs with an Ig 3 end had a marked processing advantage.
The APr-Ig demonstrated log-fold greater amounts of spliced
RNA product compared with the APr-Bcl-2 construct (Fig 5A).
To insure that this difference in RNA processing was evident at a
single-cell level, the stably transfected S194 bulk cell lines were
cloned by limiting dilution. All subclones of S194 examined confirmed
the same dichotomy in processing between constructs bearing Ig versus
Bcl-2 3 ends (Fig 6). Thus, the posttranscriptional advantage noted for the Bcl-2-Ig fusion RNA in
t(14;18)-bearing cells was reproduced in these processing constructs.
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| Fig 6.
RNA processing in oligoclonal subpopulations. S1
protection on total RNA from transfected cell lines. The probe (for
schematic, see Fig 5) is specific for the transfected exon II-intron I
sequences. The middle bands in the APr-Ig lanes correspond
to additional IVS-I start sites.
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Lineage-specific processing of the Ig 3 end.
We wished to assess whether the processing advantage conferred by the
Ig sequences was restricted to highly differentiated B-cell lineages
such as S194. Consequently, stable transfectants of the pre-B-cell
line 70z/3 were generated with both constructs. The amount of 95-nt
spliced RNA detected by the S1 assay was markedly greater for the
APr-Ig compared with the APr-Bcl-2 construct in this
pre-B-cell line as well (Fig 5B). In contrast, no considerable processing advantage was noted for the Ig 3 end when the
constructs were introduced into a non-B-lineage cell, the K562
erythroleukemia cell line (Fig 5C).
The processing advantage is conferred by the introns of the Ig heavy
chain gene.
As a first step to define the location of the processing effect
conferred by the Ig 3 end, a construct was generated
substituting an Ig heavy chain cDNA for the 3 end,
APr-IgIntrons (Fig 2). Removal of the Ig introns
eliminated the posttranscriptional advantage conferred by the complete
Ig 3 tail when assessed in S194 (Fig 5D). Moreover, substitution
of the JH6-C-1 intron by an unrelated, non-Ig intron
(the IVS II of the human -actin gene, APr-Igs intron; Fig
2) did not significantly improve processing of the fusion RNA, arguing
that the responsible mechanism is directly related to the presence of
Ig introns on the construct (Fig 5D).
To prove the validity of the constructs in reflecting the
processing of the cellular Bcl-2-IgH fusion genes, we determined the
half-life of the precursors as well as the spliced messages of the
constructs in S194. The actinomycin D half-lives of all precursors were
between 1.5 and 2.5 hours (Table 1). The
half-life of the spliced RNA could only be determined in the case of
APr-Ig, whereas the basal levels of the other RNAs were too
low. However, the half-life of APr-Ig was identical to that
of the cellular Bcl-2-IgH RNA previously measured in t(14;18) cell
lines.6 We further investigated nuclear and cytoplasmic RNA
from S194 cells transfected with APr-Bcl-2, APr-Ig, or
APr-Igintrons (Fig 7).
Whereas the amounts of precursor in the nucleus were similar, the level
of spliced RNA was higher in APr-Ig. Consequently, the
cytoplasmic spliced message was predominantly present in the cell line
containing this construct. The pattern of nuclear and cytoplasmic RNAs
was very similar to that of the cellular counterparts shown in Fig 1B,
suggesting that the constructs closely reflect the processing of Bcl-2
or Ig RNA. Interestingly, the APr-Igintrons construct
produced much less spliced message than APr-Ig, despite having the same amount of nuclear precursor, suggesting again a
processing advantage for the intron-containing construct.
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| Fig 7.
Distribution of precursor and spliced construct RNA in
the nucleus and cytoplasm of S194. A high but equal amount of
precursors is present in nuclear RNA for all three constructs
(APr-Bcl-2, APr-Ig, and APr-Igintrons),
whereas spliced RNA is predominantly present in APr-Ig
(nucleus and cytoplasm). Precursor bands in the cytoplasm represent
low-level contamination with nuclear RNA.
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| |
DISCUSSION |
Posttranscriptional processing as a novel mechanism of oncogene
deregulation.
Chromosomal translocations frequently result in the deregulation of
oncogenes through increased mRNA production. Transcriptional activation
after juxtaposition with the Ig enhancer has been described for the
c-myc and Bcl-2 proto-oncogenes.6,42 In the case of c-myc,
abrogation of an elongation block as well as a promotor shift have also
been noted.43 However the alteration of posttranscriptional processing of the Bcl-2-Ig fusion RNAs represent a novel mechanism of
oncogene deregulation.
The Bcl-2-Ig RNA product of the t(14;18) demonstrated a
posttranscriptional processing advantage when compared with normal Bcl-2 RNA. This marked difference was first noted by comparing Bcl-2
precursor RNA with final spliced mRNA products in mature B-cell lines
with and without the t(14;18). This processing difference appeared to
be specific for the Bcl-2-Ig fusion RNA, because both cell lines
processed endogenous -actin RNA in an equivalent manner. An
increased rate of newly initiated transcription of the Bcl-2-Ig fusion
gene in t(14;18) cells compared with the normal Bcl-2 gene in mature
B-cell lines lacking the translocation has been
documented.6,11 To eliminate the influence of
transcriptional differences, we generated a series of constructs to
further evaluate the processing advantage of Bcl-2-Ig fusion RNAs.
These used a heterologous -actin promoter-enhancer that conferred
equivalent rates of transcription with either the Bcl-2 or Ig 3
end. The analysis of precursor and spliced RNA products derived from
the constructs in stable cell lines confirmed the processing advantage
conferred by Ig versus Bcl-2 sequences. We believe that the constructs
closely reflect the real processing mechanism, because the
APr-Bcl-2 and APr-Ig constructs repeat the
pattern of nuclear and spliced Bcl-2 versus Bcl-2-IgH RNA in Figs 1B
and 7.
Posttranscriptional processing at the level of splicing or
nucleo-cytoplasmic transport is an established mechanism of normal gene
regulation. Examples include the regulation of HIV-1 expression by the
rev or rex proteins,23,44-46 the influenza virus
NS1,26 the polyoma early-late switch,47 the
ribosomal L1 protein of Xenopus laevis,48 and the human
c-fgr proto-oncogene.49 The ratios of Bcl-2 RNA species in
nucleus and cytoplasm indicated that splicing and/or
nucleocytoplasmic transport are enhanced in the t(14;18) cell lines.
The latter mechanism modulates expression in a number of
genes.50-53 Splicing and transport may be closely linked to
each other by the association of the ribonucleoprotein complexes with
the nuclear matrix.54-56 There is evidence that nuclear
architecture and the organization of chromosomes are tissue specific57,58 and that even two copies of the same gene can be processed differently in the same nucleus.59
Implications on Ig regulation.
In 1985, Grosschedl and Baltimore29 showed that Ig RNA
expression is regulated by at least three regions, including the VH promoter, the Ig enhancer, and intragenic sequences
lacking the enhancer. Our experiments provide evidence that intragenic sequences located downstream of the JH regions play a role
in splicing and transport. The regulated production of secretory versus
membrane forms of Ig mRNAs has also been attributed to splicing60 or the choice of polyadenylation
sites.61 We have tested both the Bcl-2 and C membrane
polyadenylation sites with the -actin promoter in B cells and found
no difference in their processing efficiency (U.J., unpublished
results). Milcarek et al62 have shown that
changes in the nuclear to cytoplasmic ratio are associated with
differential expression of secretory to membrane-specific Ig 2a
heavy chain RNA. This suggests that RNA processing may play an
important role in Ig regulation. Our data argue that these differences
in processing are directly related to the presence of the Ig introns.
It has previously been shown that insertion of a part of the C1
switch region resulted in high-level expression of human IgH in
transgenic mice, but not in transfected cell lines.63 Moreover, a splicing enhancer for Cµ has been identified in the IgM
M2 exon sequence.64 Removal of the
JH-CH as well as the C introns from our
constructs (APr-Igintrons) resulted in a dramatic
decrease in the spliced RNA species, despite the fact that the
-actin intron I is still retained to avoid completely intronless
constructs that may not express RNA at all.65 Neuberger and
Williams66 have shown that Ig expression increases with the
addition of more Ig introns, yet no specific intron was solely required. Substitution of the JH-CH intron by a
nonlymphoid intron (as in Apr-Igintron) had no
significant effect in our experiments. However, it is still possible
that substitution of all C introns will improve expression. In
addition, a complex interaction between the Ig introns may be necessary
for efficient expression, as is the case in the human triosephosphate
isomerase gene, in which upstream introns have an effect on RNA
3 end formation.67
It remains to be determined whether the presence of introns affects
only splicing or also RNA stability. Unfortunately, it was impossible
to determine the half-lives of the spliced messages except for
APr-Ig, which was identical to that of its cellular Bcl-2-IgH counterpart. However, the fact that the intronless construct (APr-Igintrons) had almost no spliced nuclear message
while having the same precursor half-life as APr-Ig argues
strongly in favor of a processing effect, because both spliced messages should look identical and have the same decay rates.
Efficient processing of the APr-Ig constructs was seen in
pre-B as well as mature B cells, indicating that this mechanism functions in various stages of B-cell differentiation.68
However, the lack of significant processing differences in the
nonlymphoid cell line K562 argues for a tissue specificity of this
mechanism.
In conclusion, posttranscriptional processing constitutes a novel
mechanism of activation that may contribute to the deregulation of
oncogenes that produce fusion mRNAs after chromosomal translocations. In particular, fusion messages that contain Ig information, such as the
LYT-10-Ca1 transcripts in the t(10;14)(q24;q32), could also be
affected. In addition, any fusion RNA that juxtaposes messages with
distinct lineage specificity may prove to have altered processing.
Moreover, our findings may have implications for the regulation of
normal Ig production, indicating a B-cell-specific, efficient pathway
for the posttranscriptional handling of Ig RNA.
| |
FOOTNOTES |
Submitted June 24, 1997;
accepted January 8, 1998.
Supported by a grant of the "Max Kade Foundation," by Grant No.
P-7565 of the Austrian "Fonds zur Foerderung der wissenschaftlichen Forschung," by grant "Kommission Onkologie" of the University of Vienna, and by National Institutes of Health Grant No. P01 CA49712-05.
Presented in part at the Second Annual Meeting of the European
Haematology Association, May 29, to June 1, 1996, in Paris, France.
Address reprint requests to Ulrich Jaeger, MD,
Klinik fuer Innere Medizin I, Haematologie 6I, Waehringer Guertel
18-20, A-1090 Vienna, Austria.
The publication costs of this article were defrayed in part by
page charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank M. Bergmann and E. Roth for their help, C. Milliman
and T. Carlisle for expert technical assistance, T. Ley for stimulating
discussions, and Roche Serena for support.
| |
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Abstract
Introduction
Abstract
