SV40 T1-mRNA trans-splicing and translation requires that the in vitro synthesized cRNA is capped before microinjection

• Published in: FEBS letters Vol. 394; no. 2; pp. 233 - 236
• Main Authors: Graessmann, M., Eul, J., Berg, B., Zimmermann, C., Graessmann, A.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 30.09.1996
• Subjects: Animals; Antigens, Polyomavirus Transforming - analysis; Antigens, Polyomavirus Transforming - genetics; Blotting, Northern; Cap structure; Cell Nucleus - metabolism; Cells, Cultured; DNA Primers; Electrophoresis, Agar Gel; Fluorescent Antibody Technique; Microinjection; Microinjections; Microscopy, Fluorescence; Nuclear export; Polymerase Chain Reaction; pre-mRNA synthesis in vitro; Protein Biosynthesis; Rats; RNA Caps; RNA Precursors - chemical synthesis; RNA Precursors - genetics; RNA Precursors - metabolism; RNA Splicing; RNA, Complementary - genetics; RNA, Complementary - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Simian virus 40 - genetics; SV40 T1-antigen; Trans-splicing in vivo; pre-mRNA synthesis in vitro; Microinjection; SV40 T1-antigen; Trans-splicing in vivo; Cap structure; Nuclear export
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(96)00956-8

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 m 7G(5′)ppp(5′)G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.