Article Text
Abstract
The LMNA gene gives rise to at least three isoforms (lamin A, C, lamin AΔ10) as a result of normal alternative splicing, regulated by cis- and trans-acting regulatory factors, as well as the 5′ and 3′ untranslated regions of the gene. The two main isoforms, lamin A and C, are constitutive components of the fibrous nuclear lamina and have diverse physiological roles, ranging from mechanical nuclear membrane maintenance to gene regulation. The clinical spectrum of diseases (called ‘laminopathies’) caused by LMNA mutations is broad, including at least eight well-characterised phenotypes, some of which are confined to the skeletal muscles or skin, while others are multisystemic. This review discusses the different alternatively spliced isoforms of LMNA and the regulation of LMNA splicing, as well as the subgroup of mutations that affect splicing of LMNA pre-mRNA, and also seeks to bridge the mis-splicing of LMNA at transcript level and the resulting clinical phenotypes. Finally, we discuss the manipulation of LMNA splicing by splice-switching antisense oligonucleotides and its therapeutic potential for the treatment of some laminopathies.
- Molecular Genetics
- Muscle Disease
- Developmental
- Neuromuscular Disease
Statistics from Altmetric.com
Introduction
The type V intermediate filament protein lamin is the main component of the nuclear lamina, which underlies the inner nuclear membrane. According to their sequence homologies, solubility during cell mitosis and expression patterns, lamin proteins are divided into A type (lamin A and C) and B type (lamin B1 and B2). Lamin B1 and B2 are encoded by two different genes (LMNB1 and LMNB2), while lamin A and C are produced through alternative splicing from the same gene (LMNA). A and B type lamins have different distribution and expression profiles. Lamin A/C is expressed only in differentiated cells and is not present in cells of neuroendocrine or haematopoietic origin.1 Lamin B2 is expressed throughout embryogenesis and is universally distributed in all cell types, whereas lamin B1 is not expressed in muscle cells and fibroblasts.1 The A and B type lamins form a distinctive inter-connected fibrous meshwork2 which provides the scaffold for the mechanical support of the nuclear membrane. The nuclear lamina participates in the linkage of the nucleo- and cytoskeleton and enables cross-talk between the nucleoplasm and cytoplasm.3 Most genes in the lamina associated chromatin regions are in a transcription repression state.4 Additionally, the lamina is involved in cell mitosis, and goes through an organised process of disassembly during prophase and reassembly in late telophase.5
To date, 464 different mutations in LMNA have been reported and are associated with a heterogeneous group of diseases, collectively known as the ‘laminopathies’ (http://www.umd.be/LMNA/). These conditions can have severe multisystemic effects or may be confined to certain tissues such as skin or skeletal muscle. Far fewer mutations, and only three associated clinical phenotypes, have been reported for LMNB1 and LMNB2.6–8 The greatest interest and the majority of studies have focused on LMNA mutations and their pathophysiological effects. Thus far, point mutations, deletions, duplications and insertion mutations have been identified in LMNA. Among these, a subgroup of point mutations have been postulated to exert their effects by interfering with the normal splicing processes, resulting in the generation of abnormal transcripts and truncated or elongated protein isoforms in affected tissues.
This review discusses the normal splicing of LMNA and its regulation, the aberrant splicing induced by disease-causing mutations, and the resulting pathophysiological disturbances in affected tissues. In addition, we discuss the manipulation of LMNA splicing using antisense oligonucleotides and the potential therapeutic significance of splice modifying techniques for the treatment of the laminopathies.
Normal LMNA isoforms
It has been suggested that alternative splicing occurs in up to 90% of human genes.9 LMNA normally encodes at least three different isoforms: lamin A, lamin C, and lamin AΔ10. A fourth isoform, lamin C2, has been found to be involved in rodent spermatogenesis, through usage of an alternative promoter and transcription initiation codon,10 and is required for chromosomal repositioning during meiosis.11 Lamin C2 is yet to be found in human tissues. Lamin AΔ10, which is translated from the LMNA mRNA as a result of exon 10 skipping,12 is present in a number of human tissues, but its function remains unknown.
The major products of LMNA, lamin A and C, arise from alternative splicing involving exon 10 (figures 1 and 2). The full-length product LMNA is processed such that all 12 exons are spliced together. However, the LMNC transcript arises from exon 10 being the last exon with 123 bases of intron 10 becoming the 3′ untranslated region (UTR). The LMNC splice form only encodes an additional six lamin C-specific amino acids downstream of that encoded by exon 10.
Lamin A consists of 12 exons and is first translated as a precursor protein, prelamin A. The last four amino acids (CSIM) of the C-terminal of prelamin A is a CAAX motif (C denotes cysteine, A an aliphatic amino acid, and X another amino acid). The C-terminal of prelamin A goes through a four-step post-translational process. First, a farnesyl group is added by farnesyltransferase to the cysteine of CAAX in the endoplasmic reticulum. Then the last three amino acids of the C-terminal (AAX) are cleaved off by Zmpste24, exposing the cysteine. Next the farnesylated cysteine is methylated by isoprenyl cysteine methyl transferase (Impt). These first three steps in prelamin A post-translational processing are conserved in proteins with the CAAX motif. Then prelamin A goes through a second endoproteolytic step, the last 15 amino acids of the C-terminal being again cleaved off by Zmpste24, producing the mature 646 amino acid lamin A. The overall processing of prelamin A to the anchoring of the mature lamin A to the lamina takes about 3 h.13 Mature lamin A and C share the first 566 amino acids, but from there on lamin A has a unique set of 80 amino acids at its C terminal and lamin C has six different amino acids.
The physiological significance of developing these post-translational modifications of lamin A during evolution is still unclear. It has been shown that all intermediate prelamin A isoforms can be imported into the nucleus, but distinct isoforms are located in different nuclear sites.14 Non-farnesylated full length prelamin A forms intranuclear foci and may be involved in recruitment of heterochromatin to the interior of nuclei, while farnesylated prelamin A is mainly present in the nuclear lamina and causes decondensation of heterochromatin.14 Paradoxically, direct synthesis of mature lamin A and bypassing of prelamin A processing in the LmnaLAO/LAO transgenic mouse results only in abnormalities of nuclear shape with no associated disease phenotype.15 Although its exact physiological role is unclear, the importance of the post-translational modification of LMNA has been illustrated by the progeroid syndromes of varying severities that involve disruption of the post-translational modification, which is discussed further on.
Lamin A/C goes through a stepwise assembly, from dimerisation, polymerisation, and assembly of the higher order proto-filaments, to formation of the filamentous nuclear lamina meshwork.16 ,17 More specifically, dimerisation involves a head-to-tail association of two neighbouring lamin monomers by overlapping of the C-terminal of the coil 2B region in the coiled-coil region between the head and tail domain, and the N-terminal of the coil 1A region.16 The dimers then form tetrameric proto-filaments.17 The nuclear membrane bound lamin A/C increases nuclear resistance to external forces, and is also important for nuclear mechano-transduction by interaction with the ‘linkers of the nucleoskeleton to the cytoskeleton’ (LINC) complex.18 ,19 The reversible phosphorylation of lamin A/C is linked to its disassembly and reassembly during cell mitosis,5 whereas the proteolysis of lamin A/C is part of the organised cell events which occur during apoptosis.19 Lamin A/C foci are also found in the nucleoplasm and are probably associated with the initiation of DNA synthesis as well as transcription.20 ,21
The relative amounts of lamin A/C vary greatly in different tissues.1 A recent study found that the stiffness of tissue matrix is positively correlated with the level of lamin A and the former is likely to be the upstream event for the latter.22 Age-dependent changes have been found in some human and animal tissues but this appears to be tissue specific. Lamin A/C transcripts increase with age in human skeletal muscle,23 ,24 but not in fibroblasts.25 On the contrary, levels of lamin A/C proteins are reduced in cardiomyocytes, osteoblasts, and chondrocytes from older mice.26 ,27 Observations in skeletal muscle samples from ageing individuals have shown that there is a shift towards increased expression of lamin A-related transcripts, and this may represent a mechanism to compensate for deteriorating nuclear function with ageing.24
It has been proposed that a B type lamin gene is the ancestral gene of the lamin family and that LMNA may have developed from this gene by the insertion of an extra exon (exon 11) during evolution.28 Interestingly, the insertion of exon 11 normally generates lamin A, but mis-splicing can lead to a product from a cryptic splice site within exon 11. This isoform, called progerin, was first identified in the context of a lethal premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). It was subsequently found that this cryptic splice site is also used at a very low levels in tissues of normal individuals and may have a physiological role in the natural course of ageing.24 ,29 Transient expression of progerin is also associated with the closure of the ductus arteriosus in neonates, suggesting that utilisation of the cryptic splice site in LMNA may also occur during tissue development.30 This finding is consistent with a previous study of the β-globin gene, showing that the cryptic splice site can be activated under normal conditions, reflecting a sub-optimal or ‘leaky’ splicing background.31
Regulation of LMNA splicing
The mRNA levels of multiple transcripts from a single gene are determined by its gene transcription and splicing efficiencies. Gene transcription is largely regulated by transcription factors, enhancer elements and promoters upstream to the transcription start site. The 5′ and 3′ UTRs at the beginning of the first and the end of the last exons, respectively, may also be involved in transcript levels by influencing mRNA stability. The recognition and splicing of an exon depends not only on the conserved sequences at splice sites, branch point and the polypyrimidine tract of intron 3′ splice site, but also on the synergic interaction of cis-acting motifs and trans-acting regulators, as well as the chromatin structure (histone epigenetic modification, nucleosome occupancy levels, GC contents).32 ,33 The regulatory motifs of a pre-mRNA include exonic splicing enhancers (ESEs)/silencers (ESSs) and intronic splicing enhancers (ISEs)/silencers (ISSs). The serine/arginine-rich splicing factors (SRSF) are the main protein family that bind to splicing enhancers and facilitate the recruitment of the spliceosome, a megadalton complex, composed of five small nuclear ribonucleoproteins (snRNPs U1, U2, U4, U5 and U6) and a series of associated proteins. The heterogeneous nuclear ribonucleoproteins (hnRNPs) primarily recognise splice silencing domains and repress splicing through a less well defined mechanism.34 Nevertheless, whether an exon is included or excluded from the mRNA does not always comply with the presence of SRSFs or hnRNP. The position, context and coordination of splice motifs all play an important part in determining the outcome of pre-mRNA processing.35 Apart from participating in the initiation of translation, protecting mRNA from exonuclease degradation, and export of mature mRNA to cytoplasm, the 5′ and 3′ UTRs are also interconnected with the splicing processes.36 ,37 The 5′ 7-methylguanosine triphosphate cap and the cap-binding complex stimulate U1 snRNP to bind to the 5′ splice site of the first intron, as well as the U6 replacement of U1 snRNP during splicing.38–40 At the 3′ end of the mRNA, the polyadenylation signal in the 3′ UTR and the poly(A) binding protein enhance the binding of the 65 kDa subunit of U2 auxillary factor (U2AF65)—a non-snRNP protein promoting the binding of U2 to intron—to the polypyrimidine tract near the 3′ splice site of the adjacent upstream intron.41
The promoter region for LMNA has been mapped to a 1.3 kb region 5′ to the translation start codon in the human.42 In the mouse, the transcription factors Sp1 and 3 can bind to a retinoic acid-responsive -75 to -36 (relative to exon 1) region within the Lmna promoter and activate the transcription of Lmna, which is repressed in undifferentiated cells.43 ,44 This promoter–retinoic acid interaction is conserved in humans.22 Upon differentiation, the structure of the chromatin around this promoter may become more accessible to transcription factors, thus activating the developmental expression of Lmna. In contrast, the germline-specific lamin C2 uses a different transcription promoter and an alternative exon 1 that are embedded in the first intron of lamin A/C. A 420 base region within Lmna intron 1 has been identified to regulate the cell type-specific expression of lamin C2.45 This region contains an AT rich motif that pairs with the hepatocyte nuclear factor-3β of the winged helix/foxhead transcription factor and another that binds the retinoic X receptor proteins.46 A brain specific microRNA, miR-9, silences the translation of Lmna in mouse brain,47 but as Lmna and Lmnc utilise different 3′ UTRs, the expression of Lmnc is not affected. In vitro studies employing neural cells differentiated from human induced pluripotent stem cells demonstrated low levels of lamin A/C compared with lamin B1, as well as a high level of miR-9.48 ,49 Nevertheless, further studies on lamin A/C and miR-9 levels in human brain tissues are needed to ascertain whether this miRNA mediated silencing of lamin A is conserved in humans. Bioinformatic studies have predicated another 10 miRNAs that may regulate the translation of LMNA by binding to the 3′ UTR,50 ,51 but this estimate needs further experimental validation. In summary, the species, tissue, and developmental specific expression of different LMNA isoforms results from an orchestrated transcription and splicing regulation from the cis-acting regulatory motifs, 5′ and 3′ UTR, and the trans-acting transcription factors and miRNAs.
Apart from the constitutive splice sites, the cryptic splice site in exon 11 is also worth mentioning, considering its implication in the development of progeroid syndromes and the subsequent targeted gene therapies. It is utilised at very low levels in non-pathological circumstances. It has been found that the splicing factor SRSF1 promotes recognition of the wild-type donor site of exon 11, while SRSF6 represses usage of the alternative splice site, probably by base pairing with the predicted stem-loop structure near the alternative splice site.52 Phosphorylated SRSF1 facilitates spliceosome assembly by recruiting U1 snRNP to the 5′ splice site.53 SRSF1 also promotes selection of the donor splice site that is more proximal to 3′ splice site,54 and may therefore lead to the preferential selection of the consensus 5′ splice site instead of the upstream alternative splice site in exon 11.
Aberrant splicing caused by mutations in LMNA
Given that LMNA is expressed in the majority of human tissues, it is of interest that mutations in LMNA tend to lead to multisystemic involvement of certain tissues, including muscle, bone, heart, blood vessel and fat, or a predominant involvement of one of these tissues. Laminopathies are a heterogeneous group of disorders which includes progeroid syndromes and lipodystrophies, as well as muscular dystrophies, cardiomyopathies and hereditary peripheral neuropathies.55 Although laminopathies can arise from a range of different types of gene mutations, for the purpose of this review we will focus on those that are caused by aberrant splicing of LMNA. Laminopathies arising from aberrant splicing demonstrate a wide range of phenotypic variability. Several factors—such as the position of the mutated functional domain, the splicing outcome of the mutation, and the expression level of the mutant protein—contribute to the disease phenotype and severity.
Progeroid syndromes
The premature ageing disease HGPS, patients with which have an average lifespan of only about 13 years and often die of premature vascular events such as myocardial infarction and stroke,56 is an example of how devastating the effects of aberrant LMNA splicing can be. In most HGPS patients, a de novo synonymous mutation (c.1824C>T) activates a cryptic splice site in exon 11 and leads to a 150 base deletion from this exon (r.1819_1968del).57 ,58 The truncated transcript (LMNA Δ150) is translated into progerin, a protein 50 amino acids shorter than the wild-type lamin A. Because of the loss of an endoproteolytic site for post-translational modification within the deleted region, progerin remains permanently farnesylated, in contrast to the transient farnesylation of prelamin A, and fails to disassociate from the nuclear membrane during mitosis. Accumulation of progerin causes large scale dysregulated gene expression,59 and eventually leads to rapid ageing in tissues such as the skin, blood vessels and heart.56 To activate the aberrant alternative splicing, the canonical HGPS mutation increases not only the putative splicing strength, but also the accessibility of the alternative splice site to the spliceosome complex, and therefore promotes splicing at the cryptic splice site.52
At least six mutations in other sites in exon 11 and the adjacent intron 11 in LMNA can also lead to HGPS or HGPS-like progeroid phenotypes. For example, de novo point mutations in exon 11 or in the first base of intron 11 can substantially inhibit usage of the wild-type donor site with or without activating the alternative splice site to a greater extent than the canonical HGPS mutation.60 ,61 Both these ‘non-canonical’ mutations cause partial abolition of lamin A production and increased progerin/lamin A ratios, and the resulting progeroid phenotypes are even more severe than the classic HGPS. The reason for the high progerin/lamin A ratios in these severe HGPS phenotypes may be that progerin protein is more stable than lamin A.60 A more recent study observed a differential allelic expression of LMNA, with the ratio of progerin/lamin A being lower if the HGPS mutation is located on the less expressed allele.62 In the case of the severe HPGS syndromes, these mutations may confer a transcription advantage to the mutant allele over the wild-type one, therefore leading to production of increased progerin and less lamin A.
Heterozygous mutations in the last nucleotide of exon 11 (c.1968G>A) or the fifth nucleotide of intron 11 (c.1968+5G>A) are also capable of inducing the 150 base deletion from exon 11, but to a lesser degree compared with the classical HGPS mutation,63 and result in the less severe atypical Werner's syndrome (AWS). In addition, it appears that HGPS can be caused not only by accumulation of progerin, but also other progerin-like proteins. In exon 11, there is yet another alternative cryptic splice site, downstream of the HGPS site, which is partially activated by a downstream C to G conversion (c.1868C>G)64 ,65 causing a 105 base deletion (r.1864_1968del) that completely overlaps with the HGPS deletion and a truncated lamin A isoform similar to progerin. The patient with accumulation of this progerin-like isoform presented with a characteristic but milder HGPS phenotype.65 It is of interest that the same heterozygous c.1968+1G>A mutation that activates the HGPS alternative splicing in the family reported by Moulson et al60 was found to cause exon 11 skipping and a fatal restrictive dermopathy phenotype in another study.66 The reason for the different splicing pattern from the same mutation is unclear. It is possible that a second defect in one of the components involved in the splicing machinery was present and altered the splicing scenario.
Collectively, mutations that cause aberrant splicing in LMNA exon 11 and accumulation of truncated farnesylated lamin A are invariably associated with progeroid disorders, and there seems to be a correlation between the disease severity and the progerin/lamin A ratio, as well as the length of the farnesylated truncated lamin A product. The shortest farnesylated lamin A due to exon 11 skipping (90 amino acid deletion) leads to the most extreme phenotype, restrictive dermopathy, while accumulation of progerin (50 amino acid deletion) leads to HGPS, and lamin A with a 35 amino acid deletion leads to a milder form of HGPS. This supports the suggestion that farnesylation of lamin A does not entirely account for the pathogeneses of these progeroid syndromes and that the deleted sequence, which encodes part of the globular tail domain, and its binding proteins also play a role in the accelerated ageing process.67
Striated muscle disorders
Aberrant splicing involving other motifs in the LMNA transcript can also cause a form of autosomal dominant limb girdle muscular dystrophy (LGMD1B). To date, at least seven LGMD1B-related mutations disrupting LMNA normal splicing have been identified, but only mutations confirmed to alter splicing outcomes are discussed here. A synonymous mutation in the last nucleotide of exon 2 (c.513G>A) causes partial retention of 45 bases of intron 2 through the activation of a downstream cryptic splice site.68 A transversion near the 5′ splice site of intron 9 (c.1608+5G>C) promotes partial retention of intron 9 and therefore introduces an extra 105 bases until it encounters a stop codon in intron 9,69 whereas a mutation near the 3′ splice site of intron 9 (c.1609-3C>G) leads to in-frame exon 10 skipping (r.1609_1698del).70 As mentioned above, lamin AΔ10 has been observed in a series of cancer cell lines as well as normal tissues,12 and its accumulation of lamin AΔ10 in the context of LGMD1B requires further investigation to ascertain its role in disease pathogenesis.
It is not only mutations in the splice sites that can disrupt normal splicing, but intronic mutations can also cause aberrant splicing. In a report of a family with predominantly heart conduction block with or without LGMD1B phenotype, a mutation in the polypyrimidine tract in intron 5 (c.937-11C>G) was found to partially activate an upstream alternative acceptor site.71 This mutation reduces the predicted splice strength of the consensus acceptor site (88 to 86) but does not change that of the cryptic splice site (http://www.umd.be/HSF/). The polypyrimidine tract is bound by U2AF and is necessary for efficient lariat formation and assembly of the spliceosome.33 Mutations in the polypyrimidine tract have been reported to cause skipping of the downstream exon or to reduce splicing efficiency without changing splicing pattern.72 ,73 The mutation may have changed an overlapping intronic splicing silencer for the upstream cryptic splice site or weakened the consensus acceptor site.
Different mutations in the last nucleotide of exon 6 (c.1157G>A, G>T) have been reported in two families with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD).74 ,75 Both mutations significantly reduce the predicted splice score (86 to 80) of the donor site. Only the c.1157G>A mutation has available transcript data for analysis, where it was reported to be transcribed into two products: one with retention of some of intron 6 with a premature stop codon in intron 6; and the other one with a normal length but a base substitution encoding lysine instead of arginine.
An A to G substitution at the 3′ of intron 3 (c.640-10A>G) created a cryptic splice site and resulted in a nine base insertion of intron 3 into LMNA transcripts, in a family with dilated cardiomyopathy (DCM) and conduction block.76 In contrast, a mutation at the 3′ splice site of intron 1 (c.357-1G>T) caused exon 2 skipping in another DCM family with arrhythmia.77
Laminopathies arising from deletions in the LMNA 5′ and 3′ UTRs
In a family with myocardial fibrosis, a deletion of the 5′ UTR as well as protein coding exon 1, including the translation start codon of LMNA, was identified.78 A shortened transcript starting from exon 2 was present despite the loss of 5′ UTR. As the amplifying primers in that study only covered the lamin A/C common exons and the mutant protein was undetectable, it is not clear if the loss of 5′ UTR results in the failure of translation initiation for the mutant isoform or this protein is not stable. It will be interesting to study whether the two 3′ UTRs for lamin A and C are utilised from the mutated allele and whether the splicing of downstream exons is influenced, as 5′ UTR is known to affect the processing of 3′ UTR as well as splicing.79 There are also reports of a shorter deletion in exon 1 that includes the translation start codon (r.-3_12del) in an EDMD family.80 Again, transcript and protein data are lacking from this study, therefore the impact of the absence of 5′ UTR on transcription, splicing and translation remains uncertain. In a family with DCM, a large deletion of exons 3 to 12 was identified.81 There was no mis-spliced isoform of LMNA or truncated lamin A protein. It appears that the deletion of 3′ UTR does not alter the splicing of the existing exon 1 and 2 in this case. Deletion of exons 3 to 12 and a complex DNA rearrangement has also been reported in a family with cardiac conduction disorder.82 Reduced immunostaining intensity and number for lamin A/C-positive nuclei in both cases implies reduced translation of lamin A/C.
Other laminopathies
In a family with the heart-hand syndrome, a point mutation in intron 9 (c.1609-12T>G) was found to activate an upstream cryptic splice site and cause partial intron 9 retention with a clinical presentation of DCM and brachydactyly.83 A severe form of Dunnigan-type familial partial lipodystrophy type 2 (FPLD2) results from a G to C transversion in intron 8 (c.1488+5G>C), which promotes intron 8 retention, leading to a disruption of the reading frame and a premature stop codon.84
To our knowledge, there have not been any reports of splice site mutations in LMNA in Charcot-Marie-Tooth disease type 2B1 or mandibuloacral dysplasia, which are also laminopathies (table 1).
Modulation of LMNA splicing
The splicing pattern of LMNA pre-mRNA can not only be altered by DNA mutations related to splice sites or motifs, but can also be modulated experimentally using antisense oligonucleotides (AOs). These are synthetic single-strand oligonucleotides, usually consisting of 15–25 bases, which are capable of switching constitutive splicing patterns of pre-mRNA by annealing to pre-mRNA and thereby affect the splicing process. Using a series of AOs with a 2′-O-methoxyethyl modified base and a phosphorothioate backbone (2′-MOEs) targeting a downstream domain of the HGPS cryptic splice site in LMNA exon 11, Fong and co-workers were able to activate utilisation of this splice site and induce overexpression of progerin in human fibroblast cultures.95 A putative ESE 34 to 56 bases downstream to the cryptic splice site that participates in exon definition was proposed. Later Luo et al used AOs with a 2′-O-methyl modified base on a phosphorothioate backbone (2OMe) as well as phosphorodiamidate morpholino oligonucleotides (PMOs) to promote recognition of the cryptic splice site in human fibroblasts as well as myogenic cells.96 Some of their AOs, especially the 2OMe AOs, also caused skipping of exon 11. One of the two most potent motifs for progerin production identified in Luo's study is close to the 3′ of the putative ESE in the study by Fong et al, and the other is 116 to 135 bases 3′ to the cryptic splice site. These two motifs may either act as enhancers for the consensus donor site or silencers for the cryptic site. The disparity in findings between the two studies reflects the different dynamics of AOs with different modifications and backbones, and suggests a tissue-specific splicing environment for LMNA. Additionally, in a zebrafish model for progeria, PMOs targeting the donor site of LMNA exon 11 activate an upstream cryptic splice site and result in deletion of the last 24 bases of exon 11.97 Splicing-switching AOs have the potential to help researchers interrogate the cis regulatory motifs in LMNA and to produce in vitro models of progeroid syndromes.
The therapeutic potential of AOs to repress aberrant splicing of LMNA in pathological conditions has also been explored. Scaffidi and co-workers used a morpholino AO that masks the HGPS mutation and the cryptic splice site to restore usage of the consensus donor site of exon 11 in HGPS fibrolasts.98 Osorio and co-workers were able to substantially inhibit splicing at the cryptic splice site in a HGPS mouse model and in HPGS fibroblasts by cocktail transfection of two PMOs, one annealing to the donor site of exon 10 for LMNA, and the other to the HGPS mutation.99 Because one PMO targeted the consensus donor site of exon 10 specific for LMNA, transcription of LMNA was abolished whereas transcription of LMNC did not appear to be affected. Finally, Huang et al designed a short hairpin RNA (shRNA) targeting the mature LMNA Δ150 mRNA in HPGS and induced RNA-induced silencing complex-mediated degradation of LMNA Δ150 mRNA without affecting wild-type LMNA and LMNC transcripts.100
Finally, there are many in vitro and in vivo models employing knock-in transgenes with specific mutations in LMNA to re-enact the aberrant splicing scenario in naturally occurring laminopathies99 that are beyond the scope of this review.
Conclusions
The normal operation of the nuclear lamina is of vital importance to a myriad of physiological processes and requires the proper transcription, co- and post-transcriptional modification, translation and post-translational processing of its main components lamin A and C. Correct splicing of LMNA is therefore intimately related with nuclear functioning. By identifying more regulatory motifs and with better understanding of the regulation of LMNA splicing, it may be possible to correct or bypass the aberrant splicing caused by pathogenic mutations. Therein lies hope of treating currently incurable laminopathies caused by aberrant splicing.
Acknowledgments
Yue-Bei Luo was supported by a China Scholarship Council-University of Western Australia joint PhD scholarship. The authors apologise to the researchers whose studies are relevant to but not cited in this review.
References
Footnotes
-
Contributors Y-BL, FM and SW all contributed to the review of literature and manuscript preparation.
-
Funding This work was supported by the Neuromuscular Foundation of Western Australia.
-
Competing interests None.
-
Provenance and peer review Commissioned; internally peer reviewed.