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 synt hesized RNA (cRNA) is trans-spliced after microinjection into the nucl ei of mammalian tissue culture cells, The template used for cRNA synth esis was the early SV40 BstXI/BamHI DNA fragment. This DNA fragment en codes exclusively for the second T-antigen exon and contains the intac t 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 splice d to the small t-antigen 3' splice site of another cRNA molecule, Form ation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysi s and DNA sequencing, Efficient tr ans-splicing required that competit ive 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 cryp tic 5' splice site was mutated.