A MUTATIONAL ANALYSIS OF THE POLYPYRIMIDINE TRACT OF INTRONS - EFFECTS OF SEQUENCE DIFFERENCES IN PYRIMIDINE TRACTS ON SPLICING

The polypyrimidine (py) tract of introns is required for efficient spl iceosome assembly and splicing of pre-mRNAs. A detailed mutational ana lysis of the py tract of an adenovirus 2 intron was carried out. Utili zing a ''precursor in pieces'' vector system, it was possible to synth esize py tract mutant pre-mRNAs that were otherwise identical. The mut ant pre-mRNAs were analyzed for in vitro splicing, for formation of sp licing complexes, and for binding to proteins in the HeLa nuclear extr act. Chimeric pre-mRNAs that contained the yeast branch point consensu s sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutati onal analysis showed the following. First, any mutation in the py trac t that affected splicing did so by interferring with complex A formati on in spliceosome assembly. Second, introduction of purines into the p y tract is detrimental only if the length of the tract is shortened an d if there is a reduction in the number of consecutive uracil residues . Third, uracil and cytosine do not have equivalent functions in the p y tract. Our results with chimeric pre-mRNAs also show that a strong p y tract can partially replace a weak branch point sequence and a stron g branch point sequence can partially replace a weak py tract. Finally , the one surprising finding obtained when examining protein binding w as that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonu cleoprotein C proteins and yet spliced close to wild type level.