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, 9 (1), 14075

Classes of Non-Conventional Tetraspanins Defined by Alternative Splicing

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Classes of Non-Conventional Tetraspanins Defined by Alternative Splicing

Nikolas Hochheimer et al. Sci Rep.

Abstract

Tetraspanins emerge as a family of membrane proteins mediating an exceptional broad diversity of functions. The naming refers to their four transmembrane segments, which define the tetraspanins' typical membrane topology. In this study, we analyzed alternative splicing of tetraspanins. Besides isoforms with four transmembrane segments, most mRNA sequences are coding for isoforms with one, two or three transmembrane segments, representing structurally mono-, di- and trispanins. Moreover, alternative splicing may alter transmembrane topology, delete parts of the large extracellular loop, or generate alternative N- or C-termini. As a result, we define structure-based classes of non-conventional tetraspanins. The increase in gene products by alternative splicing is associated with an unexpected high structural variability of tetraspanins. We speculate that non-conventional tetraspanins have roles in regulating ER exit and modulating tetraspanin-enriched microdomain function.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Conventional tetraspanin topology. Depicted is the typical topology of a tetraspanin. Intracellular domains include the N-terminus, the small intracellular loop (SIL), and the C-terminus, which are all short (for exceptions see Table S1). At the extracellular site, a small extracellular loop (SEL) connects transmembrane segment 1 (TMS1) and TMS2 and a large extracellular loop (LEL) TMS3 and TMS4. For the complete tetraspanin and its different segments, the three numbers (xx-yy-zz) indicate the sequence lengths of the shortest sequence (xx), the average sequence (yy) and the longest sequence (zz) (for details see Table S1).
Figure 2
Figure 2
Isoforms of Tspan6. (A) Top, cartoon illustrating the genomic sequence of Tspan6 as exons (white boxes) and introns (grey boxes). Exon numbering refers to the genomic sequence. From the genomic sequence, five different mRNAs derive. Here, exon numbering (green) refers to mRNA variant 1. Green boxes mark the open reading frame. Exon-skipping is indicated by leaving out the exon. When compared to splice variant 1, a shortened exon box indicates the use of an AS site. The green exon numbering is used for comparison of the respective mRNA variant 1 to the splice variants in Tab. 1. (B) Left, helical structural elements of Tspan6 predicted by Seigneuret et al.. The cartoons of the isoforms only illustrate the alterations in the primary structure and are no predictions of the protein topology. TMS, transmembrane segment; SIL, small intracellular loop; SEL, small extracellular loop; LEL, large extracellular loop; α - ε, helices of the LEL. Dashed lines mark missing parts (filled white). The asterisk in isoform 3 marks the alternative C-terminus.
Figure 3
Figure 3
Predicted topology of tetraspanin isoforms. (A) Linearized proteins. Dashed lines indicate deleted parts. Green cylinders, α-helical structure predicted by Jpred. Grey cylinders, α-helical structure not predicted by Jpred but by Seigneuret et al., and in case of CD81 revealed from crystallographic data (Kitadokoru et al., 2001). Patterned green marks the predicted transmembrane helices (TMHMM Server, 2.0). The length of the sections scales with the number of amino acids. TMS, transmembrane segment; SEL, small extracellular loop; SIL, small intracellular loop; LEL, large extracellular loop; α - ε, α-helices in the LEL. For Tspan6, no alpha helical structure of the variable domain is predicted wherefore no γ- and δ-helix are depicted. The AS of Tspan6 Iso3 leads to an alternative C-terminus. For CD82 in the variable domain only the γ-helix is predicted to be α-helical. (B) Topology of the tetraspanin isoforms illustrated in (A) with reference to the prediction which parts are intra- and extracellular (TMHMM Server, 2.0). Isoforms with an inverted topology are indicated by an asterisk. Yellow and orange spheres indicate cysteine- and glycine-residues, respectively. Cysteine-residues form disulfide bridges in the LEL; the glycine-residue is part of a conserved CCG-motif.
Figure 4
Figure 4
Classes of tetraspanins. Based on the analysis illustrated for the examples Tspan6, CD81 and CD82 (Fig. 3), all tetraspanins and their isoforms were classified as mono-, di-, tri- or tetraspanins. Subclasses result from the type of remaining TMSs, or whether a novel TMS is formed. Alteration of the N- or C- terminus, or the LEL define further subclasses. Isoforms with a partially or completely inverted topology are marked by an asterisk.
Figure 5
Figure 5
Evaluation of the expression probability of alternatively spliced mRNAs. (A) Sections of an mRNA. Cap, 5′-Cap; 5′ untranslated region (5′ UTR); ORF, open reading frame (ORF); 3′ untranslated region (3′ UTR); (A)n, poly(A) tail. (B) All alternatively spliced mRNAs lack retained introns and PTCs. In addition, mRNAs were analyzed for an upstream open reading frame (uORF), which induces NMD. They were also tested for alterations in the 3′UTR that could be associated with NMD, retention in the nucleus via nuclear RNA quality control, and miRNA-based gene silencing. Finally, they were analyzed for alteration in the 5′UTR that can alter the expression level of the mRNA. Based on these criteria, the mRNAs were sorted into three groups ranking their expression probability from very likely expressed (green - none of the criteria match), likely expressed (yellow - only alterations in the 5′UTR), or degraded (red - uORF and/or alteration in the 3′UTR).
Figure 6
Figure 6
Trafficking and possible functions of non-conventional tetraspanins. Top, illustration of alternative splicing and trafficking from the ER to the plasma membrane. Transcription of the genomic DNA (black) generates pre-mRNA with introns (blue) and exons (red). AS generates two additional different mRNAs. After translation and insertion into the ER membrane, apart from the classical pathway (middle), isoforms may behave differently in two ways. Middle, the conventional tetraspanin (green) interacts with a binding partner (orange) and both are co-transported to the plasma membrane, where the tetraspanin forms a TEM. Left, most isoforms lack TMS. The isoform shown (green) is an example from the largest group of dispanins. They cannot exit the ER, but may still interact with other proteins. Thus, if it is degraded together with the binding partner, the surface expression level of the binding partner is altered. Right, the LEL deleted isoform (green) does not interact with its binding partner (orange) but exits the ER and forms TEMs in the plasma membrane. These TEMs would lack one or more co-factors and would therefore be non-functional or differently acting TEMs. Bottom, the lower panels show confocal micrographs of GFP-labeled Tspan15 Iso2 (the conventional Tspan15 is shown in Fig. S11), CD53 or CD53 Iso2 expressed in HepG2 cells (for non-GFP-expressing control cells see Fig. S10; Western blot analysis documents the correct size of the expressed constructs; see Fig. S12). Tspan15 reaches the plasma membrane (Fig. S11), whereas Tspan15 Iso2 remains in the ER (for co-staining analysis with an ER marker see Fig. S13). Bottom, upper panels, ER retention is confirmed by analysis of cell-free plasma membrane sheets that were visualized by the membrane dye TMA-DPH. In the respective GFP-channel, only a few Tspan15 Iso2 spots are detected, that arise from ER-PM contact sites. In contrast, CD53 and CD53 Iso2 readily reach the plasma membrane, albeit CD53 Iso2 less efficient. CD53 Iso2 has lost its glycosylation sites and therefore appears in Western blot analysis as a single band (Fig. S12).

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