Abstract
rp5l B pre-mRNA, like many Saccharomyces cerevisiae primary transcripts, contains a secondary structure within its intron sequence. The structure is required for optimal in vivo splicing efficiency and includes two complementary regions near the 5' splice site and the branchpoint (UB1 and DB1, respectively). An intron-containing RNA was probed in vitro with RNase T1 and dimethyl sulfate (DMS), and is folded as expected. We have also examined in vitro splicing of rp5l B pre-mRNA, by analyzing the formation of splicing complexes as well as splicing products. Similar analyses were done with mutant pre-mRNAs containing shortened or lengthened UB1/DB1 base pairing regions. Our experiments indicate that the secondary structure acts at an early step of spliceosome assembly to aid the formation of U1 snRNP-containing commitment complexes. pre-mRNAs were probed with DMS in vivo and the folding takes place inside cells. The effect of the different UB1/DB1 interactions on in vivo splicing efficiency was also analyzed. The results are consistent with the idea that the intramolecular interaction takes place prior to or at the beginning of spliceosome assembly.
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