(Received for publication, July 24, 1995; and in revised form, January 16, 1996)
From the
Expression of the A-type lamins was studied in the lung
adenocarcinoma cell line GLC-A1. A-type lamins, consisting of lamin A
and C, are two products arising from the same gene by alternative
splicing. Northern blotting showed in GLC-A1 a relatively low
expression level of lamin C and an even lower expression level of lamin
A as compared to other adenocarcinoma cell lines. Immunofluorescence
studies revealed highly irregular nuclear inclusions of lamin A,
suggesting protein or gene expression abnormalities. Reverse
transcriptase-polymerase chain reaction-based cDNA analysis followed by
sequencing indicated the presence of an as yet unidentified alternative
splicing product of the lamin A/C gene. This product differs from lamin
A by the absence of the 5` part of exon 10 (90 nucleotides). Therefore
we propose to designate this product lamin A10. Deletion of the 30
amino acids encoded by exon 10 was predicted to result in a shift in pI
of the protein from 7.4 to approximately 8.6, which was confirmed by
two-dimensional immunoblotting. mRNA analysis in a variety of cell
lines, normal colon tissue as well as carcinomas demonstrated the
presence of lamin A
10 in all samples examined, suggesting its
presence in a variety of cell types.
Lamins are intermediate filament-type proteins which form the major components of the nuclear lamina. Two main types of lamins are known in mammals, i.e. A-type lamins and B-type lamins. The B-type lamins, B1 and B2, are encoded by two distinct genes(1) , while lamin B3 is a recently discovered alternative splicing product of the lamin B2 gene in embryonic cells(2) . At least one of the B-type lamins is ubiquitously expressed in mammalian cells and their expression appears to be independent of the state of cellular differentiation(3, 4) . The A-type lamins, represented by lamins A and C, are products arising from one gene by alternative splicing(5, 6, 7) . A-type lamin expression appears to be related to the state of cellular differentiation. In general, well differentiated cells express A-type lamins, whereas undifferentiated cells synthesize low or undetectable levels of A-type lamins(8, 9, 10) . In addition it is shown that A-type lamins are not expressed in proliferating cells of some adult tissues such as basal cells of the skin (11) or certain lineages of the hematopoietic system(12) . The recent unraveling of the complete human lamin A/C gene (7) has provided a better insight into the mechanism by which lamins A and C are generated from the same gene. The site for alternative splicing has been demonstrated to be located within exon 10. While exon 1 through 9 of the lamin A and C mRNAs are identical, lamin A mRNA further contains the 5` 90 bases of exon 10, followed by exon 11 and 12. In contrast the lamin C messenger contains the complete 111-base sequence of exon 10, but not exon 11 and 12. In this report we describe a third splicing product of the lamin A/C gene, identical to lamin A with the exception of the absence of exon 10.
Figure 2: Northern blotting of cell lines GLC-A1, GLC-A2, NL-Ac1, and NCI-H125 (lanes 1-4), hybridized with the lamin A probe (Panel A). Note the reduced expression of A-type lamins in GLC-A1 (lane 1). Panel B, lamin B1 probe; Panel C, GAPDH probe.
Figure 3:
Panel A, region of interest of the lamin A
cDNA. Numbering started from nucleotide 1. Upper numbers indicate restriction sites and exon boundaries of interest. Lower numbers correspond to first nucleotide recognized by
primers 1 through 4 (p1-p4). Numbers in boxes correspond to exon numbering according to Lin and Worman (7) . Panel B, agarose gel electrophoretic analyses of
PCR products resulting from primers 1 and 2 on cell line GLC-A1 (lane 1). Note the presence of two bands with sizes between
800 and 900 bp (double arrow). Excision and purification of
each band, followed by electrophoresis, resulted in two bands with
distinct molecular weights (lanes 2 and 3). Panel
C, restriction fragment length analysis of the normal lamin A (lanes 1 and 3) and lamin A10 (lanes 2 and 4) using SstI and NcoI (lanes 1 and 2), or PstI and SstII (lanes 3 and 4). Note that after digestion with the first set of
restriction enzymes the fragment of 704 bp (lane 1) is
shortened in lamin A
10 (lane 2, arrow), and
after digestion with the second set of restriction enzymes the expected
fragment of 211 bp is shortened by approximately 100 bp (compare lanes 3 and 4, arrow). These results
indicate a deletion between the PstI sites of exon 9 and 11. Panel D, structure of the three different mRNAs resulting from
alternative splicing of the lamin A/C gene. Panel E, schematic
diagram (adapted from Nigg(42) ) showing the impact of the
missing exon 10 in lamin A at the protein level. P,
phosphorylation site; NLS, nuclear localization signal; (E/D)
, triple repeat of Glu and Asp; H
, a sequence of four histidines; CAAX,
motif for isoprenylation of lamin A.
Figure 1: Immunofluorescence of cell line GLC-A1 (Panels A and B) and GLC-A2 (C), using an antibody to lamin A (Panels A and C) or lamin B2 (Panel B). Bar represents 20 µm.
Figure 4:
Analysis of PCR products for the presence
of lamin A and lamin A10 cDNA. PCR using primers 1 and 2 was
followed by a PCR using primers 3 and 4 in all cases, except for cell
lines LCLC-103H and NCI-H125, which were subjected to a single round of
PCR reactions using primer 3 and 4. Panel A, lamin A
10
cDNA (lane 1), normal lamin A cDNA (lane 2), GLC-A1 (lane 3), LCLC-103 (lane 4), NCI-H125 (lane
5), NCI-H23 (lane 6), normal lamin A cDNA (lane
7), GLC-A2 (lane 8), and NL-Ac1 (lane 9). Panel B, cDNA from cell line T24 (lane 1), T47D (lane 2), NCI-H460 (lane 3), MR65 (lane 4),
SK-N-SH (lane 5), normal colon (lane 6), and four
different adenocarcinomas of the lung (lanes 7-10). The upper panel shows ethidium bromide-stained agarose gels with
levels of the expected lamin A and lamin A
10 bands denoted by an arrow. Middle panel, hybridization with
P-end-labeled primer 3, hybridizing to both lamin A and
lamin A
10 cDNA. Lower panel, hybridization with
P-end-labeled oligonucleotide 5, specifically hybridizing
to lamin A
10 cDNA only. m = 100-bp ladder
markers
Figure 5: PCR-based analysis of genomic DNA of human placenta, human leukocytes, and cell lines NL-Ac1 and GLC-A1. In the first lane for each sample, primers 3 and 6 were used, and in the second lane primers 3 and 4. Arrows indicate the level of the 1404-bp band (upper) and 649-bp fragment (lower arrow). Note that, in the 100-bp ladder markers (m), bands below 600 bp are barely visible.
Figure 6:
Immunoblot detection of lamin A and lamin
A10 after two-dimensional nonequilibrium gel electrophoresis. Note
the presence of normal sized lamin A with the expected pI of around
7.0, while an additional spot with lower molecular weight and more
basic pI is also found (arrow).
In this report we describe the widespread occurrence of an as
yet unidentified splicing product of the lamin A/C gene that we
designate lamin A10, since exon 10 is absent in this transcript.
In a previous report we have demonstrated the presence of an A-type
lamin protein, which forms intranuclear aggregates in cell line
GLC-A1(24) . In contrast to normal, perinuclear A-type lamins,
these intranuclear aggregates could be largely extracted by Triton
X-100, indicating that these aggregates are not assembled into the
nuclear matrix(36) . This abnormal nuclear localization of the
protein suggested a distortion in the mRNA region coding for the
carboxyl-terminal part of the protein, since this part is known to
govern targeting to the nucleus by the nuclear localization signal, and
the CAAX-motif, the isoprenylation site of (pre)lamin A, which
is essential for a proper incorporation into the nuclear
lamina(37, 38, 39) . The structure of this
region of the mRNA was examined by RT-PCR. Sequencing showed that the
correct sequence for both motifs was present in the cDNA examined.
However, gel analysis showed the presence of an additional shortened
PCR product. Restriction fragment analysis, followed by sequencing
showed that in the otherwise normal lamin A cDNA sequence exon 10 was
lacking. Therefore we designated this novel protein lamin A
10.
Hybridization with oligonucleotides hybridizing to either lamin
A10 cDNA alone or both lamin A
10 and lamin A cDNA showed that
lamin A
10 is expressed in a variety of tissues, since all samples
examined were positive. In addition, we found that the ratio of
expression levels of lamin A
10 and lamin A varied significantly
between samples. Especially in the lung cancer cell lines the relative
concentration of the lamin A
10 can be high. Similar differences in
the expression ratio were observed between lamin A and lamin C at both
protein and mRNA level(8, 24) . Which mechanism is
involved in regulating the relative expression of the three alternative
splicing products of the lamin A/C gene, remains to be elucidated.
Preliminary studies show that different cultures from the same cell
line can express different ratios of lamin A to lamin A
10, which
might be explained by differences in cell density.
A positive
identification of the protein encoded by the lamin A10 mRNA is not
yet possible since no antibody specific for this product is available.
Therefore we cannot yet state that the abnormal lamin A expression
patterns as seen in GLC-A1 are represented by the lamin A
10
protein. However, evidence that indeed lamin A
10 mRNA is
translated into protein comes from one-dimensional (24) and
two-dimensional gel electrophoresis followed by immunoblotting studies.
Computer assisted calculation of the pI of the lamin A
10 protein
indicates a value of 8.58 as compared to a theoretical pI of 7.4 for
normal lamin A (PepStats, CAOS/CAMM, Nijmegen, The Netherlands).
Furthermore, a deletion of 30 amino acids should give rise to an
approximately 3.5-kDa smaller protein. In one-dimensional gel
electrophoresis a protein smaller than lamin A was detected with the
lamin A antibody 133A2(24, 25) . The additional
protein spot detected in two-dimensional immunoblotting is
significantly more basic (approximately 1 pI value), about 5 kDa
smaller than the normal lamin, and fulfils the predicted
electrophoretic characteristics of lamin A
10. The possibility that
lamin A
10 is a result of a translocation or deletion of lamina A/C
or is a transcript from a closely related as yet unknown gene has been
examined. A previous study suggests that such a gene might
exist(40) . Our PCR analysis within the region between exon 9
and 11 of genomic DNA provides no evidence for an additional gene.
Thus, a single gene is responsible for lamins A, A
10, and C. The
possibility that lamin A
10 is the result of a mutated lamin A/C
gene has been eliminated by our finding that lamin A
10 occurred in
all samples examined.
It is feasible that the presence of lamin
A10 mRNA has been overlooked in previous studies because of its
relatively low abundance as compared to lamins A and C expression. A
cell line with a low expression of normal lamins A and C has enabled us
to identify this lamin A
10 mRNA by RT-PCR. The same holds true for
protein analyses, in which lamin A
10 is easily overlooked,
especially because of the relatively large pI shift. Furthermore, only
in cells expressing low levels of the normal A-type lamins an aberrant
lamin can induce visible effects on the structure of the lamina.
Apparently, in GLC-A1 the concentration of lamin A
10 can reach
relatively high levels resulting in a distorted nuclear phenotype. The
lamin A
10 protein may be localized in the nuclear inclusions seen
in this cell line(24) . This would be in agreement with
transfection studies (39) which showed that different types of
nuclear distortions can be induced with constructs of lamin A
containing carboxyl-terminal deletions either starting at codon 456
(within exon 7) or starting at codon 550 (within exon 10). An altered
localization of the lamin A
10 protein is likely, since the
deletion of the first part of exon 10 results in loss of an acidic
domain (7 consecutive Glu or Asp residues) and a polyhistidine domain.
This is bound to have an impact on the interaction of lamin A
10
with other nuclear components. Although suggested, it is not yet shown
that this particular highly charged region is involved in chromatin
binding(41) . If the protein extracted by Triton X-100 from
GLC-A1 indeed represents lamin A
10, then it is possible that this
protein is not or only partially bound to the nuclear matrix and may be
involved in other intranuclear interactions.