Trans-splicing and alternative-tandem-cis-splicing: two ways by which mammalian cells generate a truncated SV40 T-antigen.

摘要

The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact small t-antigen intron. Rat cells transformed by the p14T, a construct that carries the Bst/Bam DNA fragment as a tail-to-head tandem duplication, synthesize a truncated T-antigen (T1-antigen) without having a direct equivalent at the DNA level. Formation of the T1-mRNA occurs by means of two distinct mechanisms: alternative-tandem-cis-splicing and trans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' splice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites are in an inverted order two Bst/Bam transcripts are required to generate 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 Bst/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' donor splice site and the distal segment the 3' acceptor site. This requires that the pre-mRNA not be cleaved after the RNA polymerase II has passed the polyadenylation signal of the proximal Bst/Bam DNA segment. Synthesis 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 separate 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/Bam segment of the p14T DNA (p14Tdelta-3) as a marker, destroying the recognition site for Pf/MI restriction enzyme. This deletion allowed the differentiation between the proximal and distal Bst/Bam segment. RT-PCR analysis and DNA sequencing confirmed that the p14Tdelta-3 transformed cells generate the T1-mRNA by intra- and inter-molecular RNA splicing.