Trans-complementation of the second step of pre-mRNA splicing by exogenous5 ' exons

During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate, Th e second step results in exon ligation, Previous results indicated that sev ere truncations of the 5' exon of the actin pre-mRNA result in a block to t he second splicing step in vitro in yeast extracts, leading to an accumulat ion of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RN A oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons, This reaction requires some of the cis elements and tra ns-acting factors that are required for a normal second step. There is no s trong sequence requirement for the exon 1 added in trans, but oligonucleoti des with complementarity to the U5 snRNA conserved loop perform the chase m ore efficiently, Using a dominant negative mutant of the DEAH-box ATPase Pr p16p and ATP depletion, we show that the stalled intermediate is blocked af ter the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated in to spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.