Identification of novel nesprin UTRs.

<p>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.</p

Nesprin-1 Central rod isoforms.

<p>A) Nesprin-1 isoforms p31^Nesp1, 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 p30^Nesp1 and p20^Nesp1 PCR amplified from at least one tissue examined. B) p50^Nesp1 localized to and polymerized microtubules in U2OS cells. p31^Nesp1 displayed a diffusive localization when transfected into U2OS cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040098#pone.0040098.s001" target="_blank">Figure S1A</a> for diffusive localization staining of p23^Nesp1, p12^Nesp1 and p41^Nesp1. C) p23^Nesp1 and p12^Nesp1 promoted nucleolar cap formation in HDFs while p31^Nesp1 localized to the nucleolus without causing any nucleolar disruption. D) p55^Nesp1 localized diffusively around the cytosol when transfected into U2OS cells and was detected in the kidney, spleen and peripheral blood leukocytes (PBL) by PCR.</p

Cloning and expression of novel Nesprin KASH and CH isoforms.

<p>A) Schematic representation of p53KASH^Nesp1 (Accession numberJQ754366) and p56CH^Nesp1 (Accession number JQ740783) relative to the nesprin-1 giant. B) p53KASH^Nesp1 localizes to the NE when transfected into U2OS cells. C) Nesprin-1 Flag-p56CH^Nesp1 localized to the nucleolus when transfected into U2OS cells. D) Nesprin-1 Flag-p56CH^Nesp1 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-p32CH^Nesp2 (Accession numberJQ754367) co-localized with FAK at focal adhesions when transfected into U2OS cells. F) p53KASH^Nesp1 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). p56CH^Nesp1 was detected in all cells and tissues examined whereas p32CH^Nesp2 was limited to U2OS cells, MBs and PBL.</p

UTRs identified through online databases.

<p>Table listing all potential UTRs identified through available online databases.</p

Cassette exons identified through online databases.

<p>An online scan of the EST and nucleotide databases indicated that the nesprin-1 and nesprin-2 genes underwent extensive alternative splicing and this was verified using PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040098#pone-0040098-g007" target="_blank">Figure 7A,B</a>).</p>*<p>Represents a stop codon for nesprin-1 exon 145 and nesprin-2 exons 110–113.</p

Nesprin-1 expression is highly adaptable.

<p>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.</p

Potential nesprin-1 isoforms.

<p>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 <i>Simpson and Roberts 2008</i> and are shown to scale.</p

Potential nesprin-2 isoforms.

<p>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 <i>Simpson and Roberts 2008</i> and are shown to scale.</p

Identification of nesprin-1 and nesprin-2 splicing events.

<p>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.</p

Schematic representation of the evolutionary conservation of SR units.

<p>(A) Nesprin-1. (C) Nesprin-2. Each SR unit is coloured according the percentage of conserved residues based on the alignments of vertebrate nesprin-1 and of nesprin-2, separately. The yellow star represents the invariant motif in the unstructured region. Arrows indicate N-termini of short isoforms. (B and D) Number of conservation pbs in each SR unit. Contiguous SR units with >5 pbs are coloured red for nesprin-1 (B) and for nesprin-2 (D).</p