Fig 1.
Detection of an alternative splice variant of murine Exoc3l2.
A. Gene map of murine Exoc3l2 illustrating its twelve exons, as annotated at the NCBI (XM_006540475.1) and Ensembl genome browsers (ENSMUSG00000011263). B. PCR of cDNA prepared from RNA isolated from mouse liver using the Exoc3l2-specific primers P9 and P11, and P4:5 and P11. C. PCR products of cDNA, prepared from RNA isolated from mouse organs, using the EXOC3L2 specific primers P10:11 with P11, and P4:5 with P11. Note the second bands detected at 250 bp in kidney and heart tissue in samples amplified with P4:5+11.
Fig 2.
Sanger sequencing of the alternative splice variant of murine Exoc3l2.
A. Sanger sequencing of the 818 bp product confirmed its identity as Exoc3l2. The sequence spanning the splice junction between exon 6 (E6) and exon 7 (E7) is presented in the left panel, and that spanning exon 10 (E10) and exon 11 (E11) is presented in the right panel. Above each plot five bases from the intronic sequence that is adjacent to each exons splice site are presented (i.e. i6-7 and i10-11). B. Sanger sequencing results from the second 250 bp product amplified using P4:5 with P11 and cDNA from mouse heart (H1 and H2), kidney (K1) and liver (L1) were aligned using MAFFT software to an Exoc3l2 mRNA reference sequence. The position of the novel alternative splice site between Exoc3l2 exon 6 (E6) and exon 11 (E11) was confirmed in each example. The alternatively spliced versions of E6 and E11 are colored in green. C. An example of the Sanger sequencing plots for the 250 bp alternatively spliced Exoc3l2. The novel splice junction is between an alternative 5’ donor site in exon 6 (E6) and an alternative 3’ acceptor site in exon 11 (E11). Five intronic bases adjacent to E6’ and ‘E11 are presented above the Sanger plot (i.e. i6-11). D. Gene map of murine Exoc3l2 illustrating the splicing pattern for the canonical (Exoc3l2) and alternative (Exoc3l2a) variants.
Fig 3.
Design of splice site-specific primers for Exoc3l2a.
A. A forward (Psj1) and reverse (Psj2) primer specific to the alternative splice junction between E6 and E11 of Exoc3l2 were designed. PCR of cDNA prepared from RNA isolated from mouse heart (E18.5 and P5) using Psj1 and P11 or P4:5 and Psj2 yielded single products, of predicted sizes. B. Melt curve profiles following RT-PCR of adult mouse heart cDNA (n = 5) with Psj1 and P11 (left plots) or P4:5 and Psj2 (right plots).
Fig 4.
Comparison of EXOC3L2 with the tunneling nanotube-forming M-Sec (EXOC3L3).
A. Murine Exoc3l2 has four paralogs encoding the proteins EXOC3L3 (aliases: TNFαIP2/M-Sec), EXOC3L4, EXOC3L and EXOC3. The % amino acid identity between the protein derived from each paralog and EXOC3L2 is presented in brackets. B. Gene map of murine Exoc3l2, a schematic view of the Exoc3l2 and Exoc3l2a mRNA transcripts are presented beneath the map. C. Gene map of murine M-Sec/Tnfαip2/Exoc3l3 illustrating the splicing pattern for the canonical (NM_009396.2) and a potential alternative splice variant (XM_006515792.3). D. Structural homology model of EXOC3L2 generated using SWISS-MODEL and based on the structure of murine M-Sec (PDB: 5B86)., the yellow domain would be excised from EXOC3L2a (left structure). Model of murine M-Sec/EXOC3L3, the yellow domain would be lost from the proposed slice variant of M-Sec (right structure). E. Two regions in the C-terminus of M-Sec that are functionally important for the formation of TNTs are illustrated as blue sticks on the grey helical backbone. F. EXOC3L2 amino acids with similar properties to those found in M-Sec’s region 1 and 2 are illustrated as orange sticks on the grey helical backbone. G. Alignment of the EXOC3L2 and M-Sec models. The regions illustrated in E and F are those framed with dotted lines in C and D, respectively. H. MUSCLE sequence alignment of EXOC3L2 with M-Sec. The two polybasic patches in the N-terminus of M-Sec and the aligned sequence of EXOC3L2 are framed in blue, and the positively charged amino acids in these regions are in bold blue text.