Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.
Two expression vectors were constructed that differ only in the presence (+) or absence (-) of an intervening sequence (IVS) in their 5'-untranslated leaders. Transient transfection into four mammalian cell lines resulted in higher levels of the indicator protein (CAT) from the IVS(+) vector (6 to 50-fold). Cytoplasmic RNA concentrations in 293s and HeLa cell lines corresponded directly to resultant protein levels; measurements in 293s cells of transcription initiation and elongation, steady-state total nuclear RNA, and cytoplasmic RNA stability, were equivalent for the two vectors. Surprisingly, the amount of poly(A)+ nuclear RNA was greater from the IVS(+) vector. Since this difference matches the ratio seen with polyadenylated cytoplasmic RNA, our results imply that splicing is coupled to a polyadenylation/transport pathway.
Department of Cell Genetics, Genentech, Inc., CA 94080.
M T
MT
Huang
C M
CM
Gorman
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase