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.
The second step results in exon ligation. Previous results
indicated that severe truncations of the 5′ exon
of the actin pre-mRNA result in a block to the second splicing
step in vitro in yeast extracts, leading to an accumulation
of intron-exon 2 lariat intermediates. We show that exogenous
exon 1 RNA oligonucleotides can chase these stalled intermediates
into lariat intron and spliced exons. This reaction requires
some of the cis elements and trans-acting
factors that are required for a normal second step. There
is no strong sequence requirement for the exon 1 added
in trans, but oligonucleotides with complementarity
to the U5 snRNA conserved loop perform the chase more efficiently.
Using a dominant negative mutant of the DEAH-box ATPase
Prp16p and ATP depletion, we show that the stalled intermediate
is blocked after the Prp16p-dependent step. These results
show that exogenous RNAs with various sequences but containing
no splicing signals can be incorporated into spliceosomes
and undergo RNA recombination and exon shuffling during
the second step of pre-mRNA splicing.