Figure 4
Exon 5-skipped LMP-2 splice variant and biogenesis of circLMP-2_e5 and matched cognate exon 5 skipping in LMP-2 splice variant. (a) The LMP-2 splice variant with LMP-2 exon 5-skipped was detected in EBV-positive cell lines with different latency programs in latent (UT) and lytic (T) states. The full-length gel image is shown in Fig. S6. The LMP-2 splice variant with exon 4 and 6 fused-amplicon was validated using Sanger sequencing. (b-c) Transmembrane topology prediction of (b) LMP-2A and (c) LMP-2B with or without LMP-2 exon 5-skipping using multiple prediction tools. The black dotted box indicates amino acids (position 308 to 333 and 188 to 214 for LMP-2A and LMP-2B respectively) that are encoded by exon 5 of LMP-2 whereas the black dotted line indicates the amino acid position where the exon 4 and exon 6 have joined in exon 5-skipped LMP-2A or LMP-2B. (d) Schematic representation of pcDNA3 constructs with wild type and truncated introns upstream and downstream of LMP-2 exon 5 used to test circLMP-2_e5 expression. Black solid lines and grey boxes represent introns and exon respectively, whereas light grey dotted lines indicates the deleted region of the introns. Each construct was transfected into HONE (EBV negative NPC) cells using Roche X-tremeGENE HP transfection reagent. (e) Relative expression of circLMP-2_e5 from different truncated intron constructs. Data represents mean ± SEM of four independent experiments. Significant p values [≤ 0.05 (*) and ≤ 0.01 (**)] as determined by Student’s T-test are indicated. ND not-detected.
