Figure 3

Complex alternative splicing to produce multiple Thy-ncR1 isoforms. A. Schematics of Thy-ncR1 isoform formation by alternative splicing. The polyadenylation sites are shown. The olfactory receptor gene OR10R2 and a pseudogene (OR10K1) encoded in the antisense strand are shown by the bold arrows. The exon numbers are shown below the diagram. B. The relative amounts of Thy-ncR1 isoforms. An RNase protection assay using riboprobes spanning the exon-exon junction of each isoform was conducted using total RNA from thymus (T) and Jurkat (J) cells. The negative control was yeast RNA (Y). The bands corresponding to each spliced isoform and the other isoforms in terms of each riboprobe are shown by closed and open arrows, respectively. The ratio of each isoform in thymus and Jurkat cells, which was calculated by dividing the band intensity of each isoform (closed arrow) by the sum of the band intensities of all isoforms (closed arrow + opened arrow), is shown below. C. Northern hybridization analyses to detect Thy-ncR1 isoforms. The DNA probes used are shown above. D. Additional splicing variants within exon 1. A schematic diagram of alternative splicing is shown above. RNase protection assay probe I derived from exon 1 is indicated by an arrow. The patterns obtained by RNase protection assays are shown in the lower panel with a diagram for each isoform (Ex 1a, 1b, and 1c). E. The combination formed by each exon 1 isoform spliced to other exons. RT-PCR using the exon 1 primer that detects all exon 1 isoforms, and an exon 2, 3, or 4 primer was carried out with RNA prepared from thymus (T), HPB-ALL (H), Jurkat (J), and SKW3 (S) cells. Three symbols (circle, triangle, and square) correspond to the exon 1 isoforms shown in D.