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 2h 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.
Funaguma
Shunsuke
S
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
Shimada
Toru
T
D000199:Actins / Q000235:genetics; D000818:Animals; D012831:Bombyx / Q000737:chemistry*; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry*; D005216:Fat Body / Q000737:chemistry*; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics; D012326:RNA Splicing
D000199:Actins; D002457:Cell Extracts; D012322:RNA Precursors