Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Abstract

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5′ -UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5 ′ splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein-RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.

Structure–function analysis and genetic interactions of the Luc7 subunit of the Saccharomyces cerevisiae U1 snRNP

“…As reported previously (Hossain et al 2009;Qiu et al 2012;Jacewicz et al 2015), the SUS1 transcripts in wild-type cells consisted predominantly of doubly spliced mature mRNA plus a minor component of partly spliced intermediate species (Fig. 5B).…”

“…5B). We chose SUS1 in light of our prior findings that SUS1 splicing is adversely affected by the Cbc2-Y24A mutation (Qiu et al 2012) and by Msl5 mutations that perturb the Msl5•branchpoint RNA interface (Jacewicz et al 2015). SUS1 is one of the few yeast genes that contain two introns and it is the splicing of the first intron (which has a nonconsensus 5 ′ splice site GUAUGA and a nonconsensus branchpoint sequence UACUGAC) that is selectively impaired in the absence of CBC (Hossain et al 2009).…”

“…Dissociation of U1 snRNP is triggered by the DEAD-box ATPase Prp28 (Staley and Guthrie 1999;Chen et al 2001;Jacewicz et al 2014), acting to disrupt the short U1:5 ′ SS RNA duplex or remodel protein-RNA contacts at the 5 ′ SS (or both). Traditional and systems genetic approaches, as well as structure-guided mutagenesis, have highlighted a rich network of genetically buffered functions during early spliceosome assembly in budding yeast, embracing the U1-specific snRNP proteins Mud1, Nam8, Yhc1 and Snp1, the U1 snRNA, the TMG cap, the Cbc2•Sto1 nuclear m G cap-binding complex (CBC), the DEAD-box ATPase Prp28, and the Msl5•Mud2 branchpoint-binding complex (Liao et al 1991;Abovich et al 1994;Colot et al 1996;Gottschalk et al 1998;Hausmann et al 2008;Wilmes et al 2008;Costanzo et al 2010;Chang et al 2012;Qiu et al 2012;Schwer et al 2013;Shuman 2014, 2015;Jacewicz et al 2015). This network is defined by the numerous instances in which null alleles of inessential players (e.g., Mud1, Nam8, Mud2) or benign mutations in essential factors (e.g., Yhc1, Snp1, Msl5) elicit synthetic lethal and sick phenotypes when combined with other benign mutations in the splicing machinery.…”

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

“…Genetic analyses in yeast have revealed an extensive network of buffered functions during spliceosome assembly, defined by the numerous instances in which null alleles of vegetatively inessential splicing factors, or benign mutations in essential players, elicit synthetic lethal and sick phenotypes when combined with other benign mutations in the splicing machinery (Liao et al 1991;Abovich et al 1994;Colot et al 1996;Gottschalk et al 1998;Hausmann et al 2008;Wilmes et al 2008;Costanzo et al 2010;Chang et al 2012;Qiu et al 2012;Schwer et al 2013;Shuman 2014, 2015;Jacewicz et al 2015;Agarwal et al 2016). The present collection of biologically active mutants targeted to conserved SmG, SmE, and SmF amino acids at their RNA binding sites or subunit interfaces enables us to survey genetic interactions of these three Sm ring subunits.…”

Structure–function analysis and genetic interactions of the Yhc1, SmD3, SmB, and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1

“…When structures are available, they can be exploited to program mutations with specific functional defects and then systematically test an allelic series for phenotypes per se and for synthetic genetic interactions with other spliceosome components or splicing factors. Toward this end, we have solved crystal structures of the yeast Msl5 • branchpoint RNA complex and the yeast Prp28•AMPPNP complex and have subjected these proteins to structure-guided mutagenesis (Jacewicz et al 2014(Jacewicz et al , 2015. However, there is (to our knowledge) no structure in hand for any component of the yeast U1 snRNP.…”