Reference: Schwer B, et al. (2017) Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2. RNA 23(3):420-430

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Abstract


A seven-subunit Sm protein ring assembles around specific U-rich RNA segments of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP crystal structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmD1 and SmD2 subunits. Of 18 conserved amino acids comprising their RNA-binding sites or intersubunit interfaces, only Arg88 in SmD1 and Arg97 in SmD2 were essential for growth. Tests for genetic interactions with non-Sm splicing factors identified benign mutations of SmD1 (N37A, R88K, R93A) and SmD2 (R49A, N66A, R97K, D99A) that were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and/or Msl1. Tests of 264 pairwise combinations of SmD1 and SmD2 alleles with each other and with a collection of SmG, SmE, SmF, SmB, and SmD3 alleles revealed 92 instances of inter-Sm synthetic lethality. We leveraged the Sm mutant collection to illuminate the function of the yeast Sm assembly factor Brr1 and its relationship to the metazoan Sm assembly factor Gemin2. Mutations in the adjacent SmE (K83A), SmF (K32A, F33A, R74K), SmD2 (R49A, N66A, E74A, R97K, D99A), and SmD1 (E18A, N37A) subunits-but none in the SmG, SmD3, and SmB subunits-were synthetically lethal with brr1Delta. Using complementation of brr1Delta lethality in two Sm mutant backgrounds as an in vivo assay of Brr1 activity, we identified as essential an N-terminal segment of Brr1 (amino acids 24-47) corresponding to the Gemin2 alpha1 helix that interacts with SmF and a Brr1 C-terminal peptide ((336)QKDLIE(341)) that, in Gemin2, interacts with SmD2.

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Journal Article
Authors
Schwer B, Roth AJ, Shuman S
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