Ecm2 Functions to Support U6 snRNA Structure and Dynamics during Spliceosome Catalysis

Abstract

Spliceosomes catalyze the removal of non‐coding regions, or introns, from pre‐mRNA. Assembly of the small nuclear RNA (snRNA) core of spliceosomes occurs anew on each intron via a series of carefully coordinated conformational rearrangements. However, the functional importance of many of the snRNA‐protein interactions guiding these rearrangements remain unknown. The splicing factor Ecm2 integrates into the spliceosome adjacent to the active site, where it interacts near a region of the U6 snRNA forming the catalytic RNA core of the spliceosome. By combining mutations in the U6 snRNA that perturb splicing catalysis with Ecm2 deletion in yeast, we have determined that Ecm2 functions during the chemical steps of splicing. While Ecm2 deletion or U6 mutations can be tolerated by yeast individually, we demonstrate strong synthetic interactions between these factors. We have further characterized the impact of Ecm2 deletion on splicing in vivo using a reporter pre‐mRNA. Neither Ecm2 deletion nor the U6 mutations impact splicing of pre‐mRNAs containing strong splice sites. However, both Ecm2 deletion and U6 mutation change splicing when the pre‐mRNAs contain weak, non‐consensus splice sites. Moreover, many changes observed with U6 and Ecm2 are dependent on both factors. For example, Ecm2 deletion promotes splicing of pre‐mRNAs containing a substitution at the +5 position of the 5'SS but mutation of U6 eliminates this effect. We propose that Ecm2 stabilizes U6 conformation so that it may re‐arrange to support the catalytic steps of splicing.