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

Abstract

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

Defining essential elements and genetic interactions of the yeast Lsm2–8 ring and demonstration that essentiality of Lsm2–8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24

“…Given the conservation from yeast to humans of the core Sm ring structure and the shared principle of RNA recognition via an amino acid triad present in each Sm subunit, one might have expected the Sm protein-RNA complex to be functionally acutely sensitive to perturbations of the protein-RNA interface and/or Sm-Sm subunit interface. This is clearly not the case in budding yeast, insofar as only two of 54 alanine mutations were lethal (Schwer and Shuman 2015;Schwer et al 2016Schwer et al , 2017. Lethal single-alanine mutations were confined to the RNA-binding sites of SmD1 (Arg88) and SmD2 (Arg97), thus highlighting the unique importance of their π-cation interactions with the sixth and seventh nucleobases of the Sm RNA site, respectively.…”

“…The remarkable tolerance of the seven yeast Sm proteins to mutations of the amino acids at their RNA interfaces or protein-protein interfaces suggested that the Sm ring system has built-in redundancy. To comprehensively address this issue, we conducted an "all-against-all" test of 406 different pairwise combinations of structure-guided mutations in any two of the Sm ring subunits, which unveiled a wide network of 137 intersubunit synthetic lethalities (Schwer and Shuman 2015;Schwer et al 2016Schwer et al , 2017. A key conclusion was that five of seven intact RNA-binding sites in the Sm ring do not suffice for in vivo function.…”

Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2

“…Yet, for SmF, SmE, SmG, SmD3, and SmB, as for so many components of the S. cerevisiae spliceosome, the effects of perturbing protein-RNA and protein-protein interactions were masked in an otherwise wild-type genetic background because of inherent functional redundancies of the yeast splicing machine. Our survey of genetic interactions with multiple non-Sm splicing factors showed that mutations of SmG, SmE, SmF, SmD3, and SmB consistently elicited synthetic lethality in the absence of U2 snRNP subunits Lea1 and Msl1 (Schwer and Shuman 2015;Schwer et al 2016). Sporadic synergies of specific Sm mutations were observed absent other early spliceosome components: Mud1, Nam8, Mud2, and TMG caps.…”

“…Crystal structures of the human Sm ring and its RNA interface with U1 and U4 snRNAs are known (Kambach et al 1999;Weber et al 2010;Kondo et al 2015;Li et al 2016), and recent cryo-EM studies of splicing complexes are providing similar insights into the budding and fission yeast Sm rings (Hang et al 2015;Nguyen et al 2015;Galej et al 2016;Wan et al 2016a,b;Yan et al 2016). We have married these advances in snRNP structural biology to the genetics of budding yeast to elucidate structure-function relationships and genetic interactions of the essential Sm ring subunits SmF, SmE, SmG, SmD3, and SmB (Schwer and Shuman 2015;Schwer et al 2016).…”

“…Analysis of 142 pairwise combinations of single SmG, SmE, SmF, SmB, and SmD3 RNA site mutations highlighted the built-in redundancies of the Sm ring, whereby subtracting RNA contacts of any one Sm subunit is tolerated but simultaneous mutation of the RNA binding sites of any two of the five Sm subunits tested was lethal in 45 instances (Schwer and Shuman 2015;Schwer et al 2016). These results suggested that six of seven intact RNA binding sites in the Sm ring might suffice for in vivo function, but five sites may not.…”