Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how nonessential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a nonessential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5′ splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing nonconsensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the first-step catalytic site, promoting second-step catalysis, and permitting alternate 5′ SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how non-essential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a non-essential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, another non-essential factor that promotes a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5' splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing non-consensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. It facilitates the formation and stabilization of the 1st-step catalytic site, promotes 2nd-step catalysis, and permits alternate 5' SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

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.