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. 2012;7(7):e40098.
doi: 10.1371/journal.pone.0040098. Epub 2012 Jul 2.

Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds

Dipen Rajgor  1 , Jason A Mellad, Flavia Autore, Qiuping Zhang, Catherine M Shanahan
Affiliations

Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds

Dipen Rajgor et al. PLoS One. 2012.

Abstract

Background: Nesprins (Nuclear envelope spectrin-repeat proteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.

Results: In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types.

Conclusions: These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification of novel nesprin UTRs.
A) cDNA ends identified by 3′ and 5′ RACE from Brain, Skeletal Muscle (SkeMus) and HeLa cDNA libraries. B) DNA sequencing results suggest that nesprin isoforms terminate with unique C-terminal ends absent from the giant isoforms as a result of intron retention. For example, isoforms utilising the N1-3′E90 UTR terminate with ‘AGAGYPHQ’ amino acids, giving it a unique fingerprint. Blue sequences show the coding regions of exons 90 and 91, black sequences show intronic regions and red sequence indicates a stop codon. C) Validation and tissue specificity of nesprin-1 UTRs identified on online databases and by RACE were confirmed by PCR amplification from a multiple tissue cDNA panel and DNA sequencing. Nesprin-1 PCRs were carried out when UTRs were identified on cDNA panels available at the time and are therefore organised into 3 separate sections. D) Validation and tissue specificity of nesprin-2 UTRs identified on online databases and by RACE were confirmed by PCR amplification from a multiple tissue cDNA panel and DNA sequencing. Nesprin-2 PCRs were carried out when UTRs were identified on cDNA panels available at the time and are therefore organised into 3 separate sections. Small Intestine and Peripheral Blood Lymphocytes have been abbreviated as ‘SI’ and ‘PBL’ respectively for all cDNA panels.
Figure 2
Figure 2. Potential nesprin-1 isoforms.
A) Genomic map of the nesprin-1 gene highlighting the positions of the nesprin-1 UTRs identified to date. B) Proposed nesprin-1 isoforms created by alternative transcription. SRs are numbered and shown according to the scheme of Simpson and Roberts 2008 and are shown to scale.
Figure 3
Figure 3. Potential nesprin-2 isoforms.
A) Genomic map of the nesprin-2 gene highlighting the positions of the nesprin-1 UTRs identified to date. B) Proposed nesprin-2 isoforms created by alternative transcription. SRs are numbered and shown according to the scheme of Simpson and Roberts 2008 and are shown to scale.
Figure 4
Figure 4. Cloning and expression of novel Nesprin KASH and CH isoforms.
A) Schematic representation of p53KASHNesp1 (Accession numberJQ754366) and p56CHNesp1 (Accession number JQ740783) relative to the nesprin-1 giant. B) p53KASHNesp1 localizes to the NE when transfected into U2OS cells. C) Nesprin-1 Flag-p56CHNesp1 localized to the nucleolus when transfected into U2OS cells. D) Nesprin-1 Flag-p56CHNesp1 localizes to actin stress fibres and with Focal Adhesion Kinase (FAK) at focal adhesions when transfected into Human Dermal Fibroblasts (HDFs). E) Nesprin-2 Flag-p32CHNesp2 (Accession numberJQ754367) co-localized with FAK at focal adhesions when transfected into U2OS cells. F) p53KASHNesp1 expression was not detected by PCR in U2OS, Human Dermal Fibroblasts (HDFs), Vascular Smooth Muscle Cells (VSMCs) or Myoblasts (MBs), however it was detected in the heart, spleen and peripheral blood leukocytes (PBL). p56CHNesp1 was detected in all cells and tissues examined whereas p32CHNesp2 was limited to U2OS cells, MBs and PBL.
Figure 5
Figure 5. Nesprin-1 Central rod isoforms.
A) Nesprin-1 isoforms p31Nesp1, p23 Nesp1, p12 Nesp1, p50 Nesp1, p41 Nesp1, p30 Nesp1 and p20 Nesp1 are potential variants which could be generated through alternative initiation and termination using UTRs located between exons 83 and 90. All isoforms except p30Nesp1 and p20Nesp1 PCR amplified from at least one tissue examined. B) p50Nesp1 localized to and polymerized microtubules in U2OS cells. p31Nesp1 displayed a diffusive localization when transfected into U2OS cells. See Figure S1A for diffusive localization staining of p23Nesp1, p12Nesp1 and p41Nesp1. C) p23Nesp1 and p12Nesp1 promoted nucleolar cap formation in HDFs while p31Nesp1 localized to the nucleolus without causing any nucleolar disruption. D) p55Nesp1 localized diffusively around the cytosol when transfected into U2OS cells and was detected in the kidney, spleen and peripheral blood leukocytes (PBL) by PCR.
Figure 6
Figure 6. Nesprin-1 expression is highly adaptable.
Expression levels of N1-3′E87, N1-3′E90 and nesprin-1 KASH domain were monitored post-siRNA knockdown using siRNAs targeting exons 90 and 136 of the nesprin-1 gene. As demonstrated si-136 increased expression of N1-3′E87 whereas si-90 reduced it’s expression. *p<0.01, ANOVA analysis, 95% confidence interval.
Figure 7
Figure 7. Identification of nesprin-1 and nesprin-2 splicing events.
A) PCR amplification across splice sites was carried out from cDNA isolated from U2OS cells. Splicing of exon 93 for nesprin-1 was observed as was the splicing for nesprin-2 exon 107’. B) PCR amplification across splice sites was carried out from cDNA isolated from VSMCs. Splicing of exon 93 for nesprin-1 was observed. Exon 107’ was retained in all nesprin-2 transcripts while splicing of exons 110–113 was also observed in these cells. +Represents bands with exon(s) excluded.
Figure 8
Figure 8. Generation of nesprin-1 and nesprin-2 ΔKASH variants.
A) Nesprin-1ΔKASH is generated through the removal of cassette exon 145, resulting in disruption of the KASH domain. Ectopically expressed p53ΔKASHNesp1 fails to localize to the NE in U2OS cells and is strongly concentrated within the nucleus and weakly in the cytosol. B) Nesprin-2ΔKASH1 is generated though the removal of exons 111–112 through the splicing event described in the previous section (splicing shown in red). C) Nesprin-2ΔKASH2 is generated through utilization of an alternative 3′UTR juxtaposed to exon 115. D) PCR-based tissue screen for ΔKASH variants shows that the removal of exon 145 for Nesprin-1ΔKASH is detected in a wide array of tissues. Nesprin-2ΔKASH1 splicing is detected pre-dominantly in the brain and kidney with small amounts also detected in the heart. +denotes the spliced Nesprin-2ΔKASH1 product. Nesprin-2ΔKASH2 was detected in the heart and spleen only. Peripheral Blood Leukocytes (PBL).
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References

    1. Zhang Q, Skepper JN, Yang F, Davies JD, Hegyi L, et al. Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J Cell Sci. 2001;114:4485–4498. - PubMed
    1. Zhang Q, Ragnauth CD, Skepper JN, Worth NF, Warren DT, et al. Nesprin-2 is a multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle. J Cell Sci. 2005;118:673–687. - PubMed
    1. Wilhelmsen K, Litjens SH, Kuikman I, Tshimbalanga N, Janssen H, et al. Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin. J Cell Biol. 2005;171:799–810. - PMC - PubMed
    1. Roux KJ, Crisp ML, Liu Q, Kim D, Kozlov S, et al. Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization. Proc Natl Acad Sci U S A. 2009;106:2194–2199. - PMC - PubMed
    1. Crisp M, Liu Q, Roux K, Rattner JB, Shanahan C, et al. Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol. 2006;172:41–53. - PMC - PubMed

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