Accurate pre-mRNA splicing using a nuclear extract from Bombyx mori fat body

A cell-free pre-mRNA splicing system was developed from the nuclear extract of Bombyx mori fat bodies. The synthetic pre-mRNA from the cytoplasmic actin gene of B. mori (BmA3 ) was used as a substrate for in vitro splicing. The pre-mRNA was accurately spliced in the nuclear extract prepared from the fat body. Sequencing of the amplicon by RT-PCR revealed that the spliced site in vitro was identical to the site in vivo. We found that a part of the input capped pre-mRNA was accurately spliced after 2 h of incubation. In addition, the RT-PCR analysis precisely mapped the branch point to an adenine located at 30 nucleotides before the 3′ end of the first intron in BmA3. This system was a first example that differentiated tissue in insects yielded active extracts for in vitro splicing.