Figure 4

Identification of a new IL-20R2 transcript (IL-20R2₃₀₂) via alternative splicing in mouse skin. (a) A schematic diagram of the mIL-20R2 gene locus and a predicted new mRNA transcript structure via alternative splicing (Exon 1 skipping). Primer locations for PCR and the location of the mRNA sequence identified by the 5′ RACE were as indicated. Known introns/exons from the IL-20R2₃₀₂ transcript and their encoded protein domains were as described in Figure 3, except the skipping of the Exon 1 led to a new Exon 2, which contained a different signal peptide (SS*) in-frame linked to the coding region from the original Exon 2. (b) Semi-quantitative RT-PCR analysis of the full-length IL-20R2₃₀₂ mRNA transcript in the skins of WT and IL-20R2^−/− BALB/c mice. DNA-free total RNA samples were reverse-transcribed followed by PCR using primer pairs as indicated. An upstream primer (P4) located within the Intron 1 region immediate upstream the Exon 2 and a downstream primer (P5) located in the 3′ UTR region were used in RT-PCR to confirm the existence of the predicted IL-20R2₃₀₂ transcript. As a control, IL-20R2₃₀₈-specific transcript was detected only in the WT mice by an Exon 1-specific upstream primer (P1) as shown in Figure 3 in combination with P5 primer. Both IL-20R2₃₀₂ and IL-20R2₃₀₈ transcripts were detected by RT-PCR using an upstream primer (P2) located in Exon 2 in combination with the same downstream P5 primer. Primers specific to RPL-19 transcript were used as control for equal RNA loading. (c) Mapping of the 5′ splice site junction of the IL-20R2₃₀₂ mRNA transcript by RT-PCR analysis. A series of upstream primers were designed from the Intron 1 sequence and used for RT-PCR in combination with a downstream primer located within the Exon 2 (P6). Two upstream primers (P7 and P8) were found to flank the new splice site within the Intron 1. P7 primer was able to detect the IL-20R2₃₀₂ transcription, whereas primer P8 could not. PCR amplification of the corresponding genomic DNA loci from the WT BALB/c mice served as a control for the integrity of the same primer pairs used for both RT-PCR and genomic DNA amplification. (d) Quantitative PCR analysis of IL-20R2 mRNA transcription in skin of WT and IL-20R2^−/− BALB/c mice. Primers P1 and P9 were specific to IL-20R2₃₀₈, and P10 and P9 were specific to IL-20R2₃₀₂. Samples were normalized to the housekeeping gene RPL-19. No mRNA was detectable with primers P1 and P9 in IL-20R2^−/− mice, and IL-20R2₃₀₂ was downregulated by over threefold in IL-20R2^−/− mice in comparison with WT mice using primers P10 and P9. Statistical significance of differences in IL-20R2 mRNA fold was analyzed using the T test. Values of P<0.01 (*) were considered statistically significant.