Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Masahiko Ajiro; Shuang Tang; John Doorbar; Zhi-Ming Zheng

doi:10.1128/JVI.00965-16

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Construction of the in vitro splicing system for the major HPV18 alternative splicing sites. (A) Diagrams of the pre-mRNAs tested for in vitro splicing assays. A U1 binding motif of 11 nt (gray boxes) was attached to the 3′ ends of pre-mRNAs 2, 4, 6, and 8 to enhance in vitro splicing. Pre-mRNAs 3 to 8 have a truncated intron ∼350 nt in size for in vitro splicing assays. (B) Splicing gels from in vitro splicing assays. The splicing reactions were performed by incubating each ³²P-labeled HPV18 pre-mRNA in panel A with HeLa nuclear extract at 30°C for 0, 1, and 2 h, and the splicing products were resolved on a 6% denaturing PAGE gel. The identities of unspliced pre-mRNA (a) and individual spliced products (b, b′, b″, and c) are indicated. The splicing efficiency (% spliced) was calculated from the splicing gels as described previously (32).