FIG. 2.

Inserting an extra copy of TPI intron 6 into TPI exon 7 moves the boundary between nonsense codons that do and do not reduce TPI mRNA abundance to the new penultimate exon, indicating that the intron functions in a context-dependent manner to position the boundary. NIH 3T3 cells were transfected, and total-cell RNA was purified and analyzed as described in the legend to Fig. 1. Transfecting plasmids consisted of the reference pMT-Gl construct and the pMT-TPI construct specified above each lane. Constructs with an extra (XS) copy of TPI intron 6 harbored the XS intron at the EcoRI site of TPI exon 7, which is designated with an asterisk in the diagram of the MT-TPI gene. The normalized value for MT-TPI mRNA harboring 189Ter and the usual intron arrangement was then calculated as a percentage of MT-TPI^Norm mRNA (Norm), which was considered to be 100. Similarly, normalized values for MT-TPI mRNAs that derived from constructs harboring two copies of intron 6 were calculated as a percentage of MT-TPI^{Norm, XS TPI intron 6} mRNA (Norm, XS TPI intron 6). Values are representative of three independently performed experiments and did not differ by more than 6%. Notably, both copies of intron 6 were efficiently and accurately spliced from pre-mRNA as indicated by sequencing products of reverse transcriptase-PCR products that extended from exon 6 through exon 7 (data not shown).