Saccharomyces cerevisiae Ecm2 modulates the catalytic steps of pre-mRNA splicing

Abstract

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.