title: Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2.
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 inSaccharomyces 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 withbrr1Δ. Using complementation ofbrr1Δ 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 α1 helix that interacts with SmF and a Brr1 C-terminal peptide (336QKDLIE341) that, in Gemin2, interacts with SmD2.
authors.affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, New York 10065, USA bschwer@med.cornell.edu s-shuman@ski.mskcc.org.
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
authors.affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Allen J
authors.initials: AJ
authors.lastname: Roth
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA bschwer@med.cornell.edu s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015870:Gene Expression; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D006801:Humans; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism
chemical_list: C102201:BRR1 protein, S cerevisiae; C108381:GEMIN2 protein, human; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins