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Supplementary Information
The construction of the knock-in LmnaL530P/L530P mice is summarized in Figure 1A (see published
text for description). The nucleotide changes introduced to alter residue L530 are shown in
Figure 1B. Adjacent to the L530P mutation, two additional nucleotide changes at the wobble
position were introduced to create a nearby AseI site. The mutated fragment was verified by
sequence analysis and swapped for its wild type counterpart in a 9.7 kb ApaI/NheI fragment
encompassing exons 3-11 of the mouse Lmna gene1. A neomycin selectable marker flanked by
loxP sites was inserted in reverse orientation to Lmna at the SmaI site of intron 9. Recombinant
ES clones were screened by Southern analysis of genomic DNA restricted with EcoRI and
probed with a 1.3 kb ApaI fragment containing intron 2 of Lmna.
The introduction of the L530P mutation in Lmna resulted in decreased levels of lamin
transcripts (Figure 2A, B), suggesting the mutation caused instability of Lmna messages. A
broad RNA band near the position of lamin C suggested that more than two spliced forms of
Lmna might be present. RT-PCR of polyA+ RNA isolated from LmnaL530P/L530P PMEFs using
primers specific for lamin C yielded two distinct bands, confirming a splicing defect near exon 9
in the mutants (Figure 2C). The RT-PCR products were subcloned, and sequence analysis
identified fragments of 471 and 212 nucleotides (nt). The 471 nt fragment contained a complete
exon 9 with the L530P point mutation, but showed incorrect splicing from exon 9 to exon 10.
The result of this splicing anomaly was that part of intron 9 was included in the message,
encoding an additional 19 amino acids in frame after exon 9 before a stop codon in the intron
was encountered (Figure 2D). The 212 nt RT-PCR fragment lacked exon 9 altogether, splicing
exon 8 directly to exon 10. Loss of exon 9 or addition of heterologous residues and loss of exon
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10 due to a premature stop codon might render lamin C products unstable, a possibility
consistent with Western analysis which showed no evidence of lamin C. Alternatively, the
putative truncated form of lamin C may be immediately tagged for degradation as an improperly
folded protein, which would be consistent with loss of lamin C at the NE and gain of cytoplasmic
lamin C-reactive peptides observed in immunofluorescence. The antibody to lamin C only
recognizes proteins containing the six amino acids specific for the lamin C product, and would
not recognize proteins translated from a transcript containing the splicing defect defined by the
471 nucleotide RT-PCR product. Splicing of lamin A transcripts was affected in a similar
manner, resulting in two analogous RT-PCR fragments showing the same loss of exon 9 or
inclusion of intron 9 sequences with an in-frame stop codon. The same splicing defects were
seen in both ES cell lines used to generate mice.
The disruption of splicing may explain why muscular dystrophy was not observed in
mice carrying L530P, a point mutation associated with AD-EDMD in humans. Whether the
L530P mutation in LMNA causes similar splicing defects in humans with AD-EDMD is unclear.
Proteins translated from mutant lamin A and C cDNAs and overexpressed in vitro with the single
amino acid change, L530P, are stable and competent to assemble into the nuclear lamina, even
though the nuclear lamina containing L530P-A-type lamins shows slight morphological
anomalies2. The L530P variant of lamin A was, however, unable to interact in vitro with
emerin2, a nuclear envelope protein associated with the X-linked form of EDMD3. The
redistribution of emerin to the cytoplasm due to a lost interaction with mutant A-type lamins may
be key in the development of muscular dystrophy. Taken together, these results suggest that the
expression of mutant A-type lamins containing the L530P variation may result in AD-EDMD,
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but a splicing defect arising from alterations in the coding sequence in exon 9 or intron 9 and
subsequent loss of lamin C from the NE results in progeria.
1.
Sullivan, T. et al. Loss of A-type lamin expression compromises nuclear envelope
integrity leading to muscular dystrophy. J Cell Biol 147, 913-20 (1999).
2.
Raharjo, W. H., Enarson, P., Sullivan, T., Stewart, C. L. & Burke, B. Nuclear envelope
defects associated with LMNA mutations cause dilated cardiomyopathy and EmeryDreifuss muscular dystrophy. J Cell Sci 114, 4447-57. (2001).
3.
Bione, S. et al. Identification of a novel X-linked gene responsible for Emery-Dreifuss
muscular dystrophy. Nature Genet. 8, 323-327 (1994).
Figure 1 Strategy of the LmnaL530P/L530P targeting construct. A The genomic fragments used to
build a construct containing the nucleotide change introducing a L530P substitution are shown.
B The nucleotide sequences of wild type (WT) Lmna and L530P mutant (MUT) Lmna after
mutagenesis are shown.
Figure 2 The mutation encoding an L530P change in A-type lamins destabilizes Lmna
transcripts causing aberrant splicing. A Poly A+ RNA was isolated from wild type (WT),
LmnaL530P/+ (HET) and LmnaL530P/L530P (MUT) PMEFs and probed with a 400 bp HindIII
fragment isolated from the rod domain of the human LMNA cDNA. The blot was re-probed
with a human actin probe (Clontech) as a loading control. B Phosphor imaging quantitation of
Lmna transcript levels. C RT-PCR was performed using a 5' primer from exon 8 and a 3'
primer from the C-specific region of exon 10. The DNA markers are the 1 Kb-Plus ladder
(Invitrogen). D Sequence analysis defined the splicing defects resulting in the 212 nt and 471 nt
RT-PCR fragments (see text). The exons shared between lamin A and lamin C proteins are in
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green, and the lamin C-specific region of exon 10 is in purple. The tan box (int) represents the
portion of intron 9 which is not spliced out of the 471 nt PCR product. The rest of the intron is
missing from that transcript. Introns and exons are not to scale.