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In vitro synthesized SV40 cRNA is trans ‐spliced after microinjection into the nuclei of mammalian cells
Author(s) -
Eul J.,
Graessmann M.,
Graessmann A.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00957-x
Subject(s) - rna splicing , microinjection , exon , microbiology and biotechnology , intron , trans splicing , messenger rna , biology , rna , splice , splice site mutation , in vitro , dna , gene , genetics
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 Bst XI/ Bam HI 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.