Figure 5

Exon 3 splicing of TREM2 is a determinant of its protein expression. (A) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. (B) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. (C) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. (D) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. (E) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t-test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. S8.