The stereochemical course of the first step of pre-mRNA splicing.
We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.
Maschhoff
K L
KL
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235.
Padgett
R A
RA
D000260:Adenoviruses, Human / Q000235:genetics; D001483:Base Sequence; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009694:Nucleic Acid Precursors / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism* / Q000648:ultrastructure; D012367:RNA, Viral / Q000378:metabolism; D013237:Stereoisomerism; D013455:Sulfur / Q000737:chemistry
D009694:Nucleic Acid Precursors; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D013455:Sulfur