In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract.
An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.
Sakamoto
H
H
Department of Biophysics, Faculty of Science, Kyoto University.
Ohno
M
M
Yasuda
K
K
Mizumoto
K
K
Shimura
Y
Y
D000818:Animals; D001345:Autoradiography; D002645:Chickens; D002855:Chromatography, Thin Layer; D003459:Crystallins / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D010957:Plasmids; D012314:RNA Cap Analogs / Q000235:genetics; D012315:RNA Caps / Q000235:genetics; D012322:RNA Precursors / Q000096:biosynthesis / Q000235:genetics*; D012326:RNA Splicing; D014158:Transcription, Genetic
D003459:Crystallins; D004274:DNA, Recombinant; D012314:RNA Cap Analogs; D012315:RNA Caps; D012322:RNA Precursors; D004247:DNA