In vitro synthesized SV40 cRNA is trans-spliced after microinjection into the nuclei of mammalian cells.
We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXl/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (T1-mRNA) by trans-splicing, the cells utilize a 5' cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3' splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5' splice site. The T1-mRNA was not generated when the cryptic 5' splice site was mutated.
Eul
J
J
Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Germany.
Graessmann
M
M
Graessmann
A
A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005091:Exons / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D008845:Microinjections; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012326:RNA Splicing; D018075:RNA, Complementary / Q000138:chemical synthesis / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D018075:RNA, Complementary; D012367:RNA, Viral