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. 2019 Jan-Dec:15:1744806919874557.
doi: 10.1177/1744806919874557.

Expression of a novel versican variant in dorsal root ganglia from spared nerve injury rats

Oliver Bogen  1   2 , Olaf Bender  1 , Pedro Alvarez  3 , Marie Kern  3 , Stefan Tomiuk  4 , Ferdinand Hucho  1 , Jon D Levine  2   3
Affiliations

Expression of a novel versican variant in dorsal root ganglia from spared nerve injury rats

Oliver Bogen et al. Mol Pain. 2019 Jan-Dec.

Abstract

The size and modular structure of versican and its gene suggest the existence of multiple splice variants. We have identified, cloned, and sequenced a previously unknown exon located within the noncoding gene sequence downstream of exon 8. This exon, which we have named exon 8β, specifies two stop-codons. mRNAs of the versican gene with exon 8β are predicted to be constitutively degraded by nonsense-mediated RNA decay. Here, we tested the hypothesis that these transcripts become expressed in a model of neuropathic pain.

Keywords: Isolectin B4; Versican; eIF2α(PO); nerve injury; neuropathic pain; nonsense mediated RNA decay.

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Figures

Figure 1.
Figure 1.
Schematic representation of the genomic structure of the versican gene, including exon structure of the five known splice variants. Its 15 exons are represented as colored rectangles. Exons 1 to 6 and 9 to 15 are represented by red rectangles. Exons 7 and 8 are represented by green rectangles. The part of exon 8 that is expressed in versican V4 is represented by a blue rectangle. The black line between the exons in the top illustration represents non-coding intervening sequences. The black lines that connect the exons in the representation of the different splice variants illustrate the splicing process through which the different versican variants are generated. Note that the start-codon for versican biosynthesis is located within exon 2. GAG: glycosaminoglycan.
Figure 2.
Figure 2.
The EST 603032745F1/exon 8β is part of a V0- and/or V1-based mRNA. (a) 2 RT-PCRs were performed to define the spatial dimensions of exon 8β; a RT-PCR with primers located within exon 8 and exon 8β amplified a 372bp DNA-fragment (lane 2), and a RT-PCR with primers located within exon 8β and exon 11 amplified a 360bp DNA-fragment (lane 3). A control PCR with primers located within exons 4 and 6 [size = 435bp]—which should detect all currently known versican splice variants—was performed to verify the quality of the RNA/cDNA preparation (lane 1). (b) Genomic context of exon 8β. Coding sequences (exons) are shown in bold letters. Non-coding, intervening sequences (introns) are shown in plain letters. Exon 8β is highlighted in blue letters. The primer sequences that were used for the amplification of cDNA-fragments including exon 8 are underlined; the top two primers were used to define the 5′-end of exon 8β, the bottom two primers were used to define its 3′-end. Of note, the two primers located within exon 8β are complementary to each other and of reverse orientation. (c) C-terminal amino acid sequence of the corresponding versican splice variant V5 (and a hypothetical variant V6). Two stop-codons (red asterisks) within the reading frame of exon 8β would terminate protein translation of the corresponding mRNA transcript downstream of exon 8, versicans GAGβ domain.
Figure 3.
Figure 3.
A novel Vcan variant is expressed in SNI rats. (a) A novel Vcan variant is transcribed in SNI but not sham control rats. PCRs on cDNA from rat DRG demonstrates that the new Vcan variant is also transcribed in spared nerve injury rats but that it is absent in sham-operated rats. 1: Vcan_exon4_for and Vcan_exon6_rev = 435bp; 2: Vcan_exon8_for and Vcan_exon8β_rev = 372bp; 3: Vcan_exon8β_for and Vcan_exon11_rev = 360bp. (b) ISR is activated in SNI rats. Analyzing the post-translational modification of eIF2α by Western blotting demonstrates a significant increase of eIF2αPO42− in DRG extracts from rats submitted to SNI compared to sham-surgery rats [eIF2αPO42− immunoractivity in SNI = 126 ± 12 arbitrary units; eIF2αPO42− immunoractivity in sham controls = 98 ± 10 arbitrary units; normalized to the reference protein eIF2α; P<0.05 (unpaired student’s t-test), a 28.4 ± 15.8% increase in the eIF2αPO42− immunoreactivity, N=9]. Note that the calculated molecular weight of eIF2α ± PO42− is 36 kDa (according to the UniProtKB database entry P68101). *P<0.05. (c) SNI rats express a C-terminally truncated Vcan variant. DRG extracts from SNI rats were analyzed by Western blotting using a set of four different antibodies, each of which is directed against one of versican’s four major structural and functional domains. Rat V2 is made of 1601 amino acids with a calculated molecular weight of ∼176 kDa. The discrepancy between its calculated and apparent molecular weight on the Western blot can be explained by its highly acidic character, which interferes with micelle formation during gel electrophoresis., The Vcan variant above V2 is composed of Vcan’s N-terminus and only the GAGβ domain. It’s domain composition and apparent molecular weight suggests that it is the C-terminal truncated Vcan variant predicted by the translation of V1-derived mRNAs carrying exon 8β.
Figure 4.
Figure 4.
Schematic representation of the potential structure of the novel versican variant V5 and its structural differences from two of the “classic” splice variants, V0 and V3. Thick black lines represent the protein backbone. Thin blue lines represent the chondroitin sulfate side chains, which are attached to versican’s GAG domains, and the thick blue line represents hyaluronan. Versican V0 is comprised of three distinct structural domains, a hyaluronan-binding domain at its N-terminus, two GAG domains in the middle portion of the protein, and a selectin-like domain at its C-terminus. V3 lacks both GAG domains and therefore comprises only the N-terminal hyaluronan-binding domain and the C-terminal selectin-like domain. V5 lacks the C-terminal selectin-like domain and therefore would only be comprised of the N-terminal hyaluronan-binding domain and the GAGβ domain.
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