TRANSSPLICING AND ALTERNATIVE-TANDEM-CIS-SPLICING - 2 WAYS BY WHICH MAMMALIAN-CELLS GENERATE A TRUNCATED SV40 T-ANTIGEN

The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact sma ll t-antigen intron. Rat cells transformed by the p14T, a construct th at carries the Bst/Bam DNA fragment as a tail-to-head tandem duplicati on, synthesize a truncated T-antigen (T1-antigen) without having a dir ect equivalent at the DNA level. Formation of the T1-mRNA occurs by me ans of two distinct mechanisms: alternative-tandem-cis-splicing and tr ans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' s plice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites a re in an inverted order two Bst/Bam transcripts are required to genera te one T1-mRNA molecule. For alternative-tandem-cis-splicing the cells utilize a 4.4 kb pre-mRNA that contains the sequence of the entire Bs t/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' dono r splice site and the distal segment the 3' acceptor site. This requir es that the pre-mRNA not be cleaved after the RNA polymerase II has pa ssed the polyadenylation signal of the proximal Bst/Bam DNA segment. S ynthesis of the 4.4 kb pre-mRNA was demonstrable by RT-PCR but not by Northern blot analysis. For trans-splicing, the cells utilize two sepa rate pre-mRNA molecules. One transcript provides the cryptic 5' splice donor site and the other the 3' splice acceptor site. To demonstrate this a three base pair deletion was introduced into the proximal Bst/B am segment of the p14T DNA (p14T Delta-3) as a marker, destroying the recognition site for Pf/MI restriction enzyme. This deletion allowed t he differentiation between the proximal and distal Bst/Bam segment. RT -PCR analysis and DNA sequencing confirmed that the p14T Delta-3 trans formed cells generate the T1-mRNA by intra- and inter-molecular RNA sp licing.