SV40 T1-mRNA trans-splicing and translation requires that the in vitro synthesized cRNA is capped before microinjection.
The purpose of this investigation was to study the effect on cap structure for trans-splicing in mammalian cells. The early SV40 Bst/Bam pre-mRNA (cRNA) was synthesized in vitro in both capped (cap-Bst/Bam-cRNA) and non-capped (Bst/Bam-cRNA) versions and microinjected into the nuclei of TC7 cells. Trans-splicing was monitored by immunofluorescence staining (T1-antigen) and by RT-PCR analysis. Cap-Bst/Bam-cRNA was trans-spliced with high efficiency, but not the Bst/Bam-cRNA molecules. Northern blot analysis revealed that both the capped and uncapped cRNA molecules had similar stability in the microinjected cells. The coinjected m7G(5')ppp(5')G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.
Graessmann
M
M
Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Germany.
Eul
J
J
Berg
B
B
Zimmermann
C
C
Graessmann
A
A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics*; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005455:Fluorescent Antibody Technique; D008845:Microinjections; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012315:RNA Caps; D012322:RNA Precursors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D012315:RNA Caps; D012322:RNA Precursors; D018075:RNA, Complementary; D012367:RNA, Viral