Correct in vivo splicing of the mouse immunoglobulin kappa light-chain pre-mRNA is dependent on 5' splice-site position even in the absence of transcription.
In transcripts from the rearranged mouse immunoglobulin kappa light-chain locus, the intron separating the variable (V) plus joining (J) exon from the constant (C) exon contains up to three additional J regions, each with a functional 5' splice site. Previously, HeLa cells transfected with DNA encoding kappa light chains have been shown to mimic kappa-producing lymphocytes in splicing exclusively to the upstream-most 5' splice site, whereas selectivity is lost when kappa transcripts containing two more J regions are incubated in HeLa cell or lymphocyte nuclear extracts. Here we demonstrate that the fidelity of in vivo splicing depends on neither V-J rearrangement, the instability of erroneously splicing transcripts, nor a hierarchy of J-region 5' splice site utilization. Analysis of the splicing of presynthesized kappa transcripts injected into Xenopus oocytes demonstrates the correct 5' splice-site selection is independent of transcription. Implications for in vitro studies of regulated splice-site pairing are discussed.
Kedes
D H
DH
Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Steitz
J A
JA
D000818:Animals; D004274:DNA, Recombinant; D006367:HeLa Cells; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000502:physiology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D008845:Microinjections; D008957:Models, Genetic; D012313:RNA / Q000302:isolation & purification; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics
D004274:DNA, Recombinant; D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D012322:RNA Precursors; D012333:RNA, Messenger; D012313:RNA