Figure 3. 54-bp insertion changed RNA alternative splicing patterns.

(A) RNA structure of insertion and varied alternative splicing sites compared between insertion and deletion types. (B) RNA expression levels of fragments near the insertion region were tested in the birds with the insertion genotype. (C) RNA expression levels of fragments near the insertion region were tested in the birds with the deletion genotype. (D) Alternative splicing in the insertion region was performed by using a reporter gene system. A 106-bp fragment including the stem-loop insertion, or normal 52-bp fragments, were cloned into the 3′UTR of the reporter gene (luciferase) followed with polyA signals. The recombined vectors were co-transfected with pRL-TK control vector into DF-1 chicken embryo fibroblast cells for 36 h to detect the firefly luciferase activity. (E) The effect of insertion mutation on mature miRNA expression in vitro. A 1300-bp fragment of the primary transcript of miR-15a-16 was cloned into the pcDNA3.1 vector (pcDNA-II and pcDNA-DD) to overexpress miRNA in DF-1 chicken embryo fibroblast cells. Mature miRNAs were tested by q-PCR. In all panels, data are presented as mean ± SD. **p < 0.01 was estimated by Student’s t-test (n = 3).