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

Abstract

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5' splice site (5'SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5'SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.

Keywords: 5′; Prp28; Sm proteins; splice site; spliceosome assembly.

FIGURE 1.

Structure-guided mutational analysis of Yhc1. (A) Alignment of the primary structures of the N-terminal domains of S. cerevisiae Yhc1 and human U1-C. Positions of side-chain identity/similarity are indicated by • above the alignment. The secondary structure elements are depicted below the alignment, with β strands as magenta arrows and α helices as cyan cylinders. U1-C/Yhc1 amino acids that coordinate zinc, make contacts to other U1 snRNP subunits, and contact the U1 snRNA or the mRNA 5′ splice site sequence are highlighted in color-coded boxes as indicated. (B) Summary of genetic interactions of the indicated YHC1-Ala alleles. (C) Prp28 bypass. Yeast prp28▵ yhc1▵ cells harboring the indicated YHC1 allele on a CEN LEU2 plasmid and either wild-type PRP28 (CEN HIS3) or an empty CEN HIS3 plasmid (prp28▵) were spot-tested for growth on YPD agar at the temperatures specified. (D) Stereo view of the human U1 snRNP structure highlighting the interactions of U1-C/Yhc1 (depicted as a cartoon trace with magenta β strands and cyan helices, with selected amino acids as stick models with beige carbons) with the RNA duplex formed by the U1 snRNA 5′ leader (5′-ACUUAC⁸C⁹U¹⁰, depicted as a stick model with gray carbons) and the mRNA 5′SS exon–intron junction (5′-A⁻²G⁻¹/G¹U², depicted as a stick model with yellow carbons). Protein–RNA contacts are indicated by dashed lines: black for hydrogen bonds and green for van der Waals interactions.

FIGURE 2.

Structure-guided mutagenesis of SmD3. (A) Stereo view of the human U1 snRNP structure highlighting the fold of SmD3 (depicted as a cartoon trace with magenta β strands and cyan helices) and its interactions with neighboring subunits U1-C/Yhc1 (green) and U1-70K/Snp1 (blue) and with the Sm site in U1 snRNA. The zinc atom nucleating the U1-C/Yhc1 fold is depicted as a magenta sphere. Selected amino acids are shown as stick models and numbered according to their positions in the yeast polypeptides. Atomic contacts are indicated by dashed lines. (B) Alignment of the primary structures of the S. cerevisiae (Sce) and human (Hsa) SmD3. Positions of side-chain identity/similarity are indicated by • above the alignment. The secondary structure elements are depicted above the alignment, with β strands as magenta arrows and α helices as cyan cylinders. SmD3 amino acids that make contacts to other U1 snRNP subunits or contact the U1 snRNA are highlighted in color-coded boxes as indicated. Reverse arrowheads indicate the boundaries of the C terminal truncations of yeast SmD3; black and red arrowheads denote viable and lethal truncations, respectively. (C) The wild-type and truncated SMD3 alleles were tested for activity by plasmid shuffle in smd3▵, smd3▵ mud2▵, smd3▵ nam8▵, smd3▵ mud1▵, and smd3▵ tgs1▵ strains. The viable FOA-resistant smd3▵ strains bearing the indicated SMD3 alleles were spot-tested for growth on YPD agar at the temperatures specified. SMD3 alleles listed at the bottom of each panel failed to complement smd3▵ in the plasmid shuffle assay and were deemed lethal in that genetic background.

FIGURE 3.

Synthetic interactions of SmD3-Ala mutants. Yeast smd3▵ strains bearing the indicated SMD3-Ala alleles on a CEN LEU2 plasmid in an otherwise wild-type (top panel), mud2▵, mud1▵, or nam8▵ background as indicated were spot-tested for growth on YPD agar at the temperatures specified. Synthetic growth defects are denoted by •.

FIGURE 4.

Synthetic interactions of SmD3-Ala mutants. Yeast smd3▵ strains bearing the indicated SMD3-Ala alleles on a CEN LEU2 plasmid in an otherwise wild-type (top panel), mud2▵, mud1▵, nam8▵, or tgs1▵ background as indicated were spot-tested for growth on YPD agar at the temperatures specified. Synthetic growth defects are denoted by •.

FIGURE 5.

Structure-guided mutagenesis of SmB. (A) Stereo view of the human U1 snRNP structure highlighting the fold of SmB (depicted as a cartoon trace with magenta β strands and cyan helices) and its interactions with neighboring subunits SmD3 (yellow) and U1-70K/Snp1 (blue) and with the Sm site in U1 snRNA. Selected amino acids are shown as stick models and numbered according to their positions in the yeast polypeptides. Atomic contacts are indicated by dashed lines. (B) Alignment of the primary structures of the S. cerevisiae (Sce), Candida glabrata (Cgl), and human (Hsa) SmB. Positions of side-chain identity/similarity in the amino-terminal segments of all three proteins are indicated by • above the alignment. Positions of side chain identity/similarity between the carboxy-terminal segments of SceSmB and CglSmB are denoted by /. The secondary structure elements of the amino-terminal domain are depicted below the alignment, with β strands as magenta arrows and α helices as cyan cylinders. SmB amino acids that make contacts to other U1 snRNP subunits and contact the U1 snRNA are highlighted in color-coded boxes as indicated. Reverse arrowheads indicate the boundaries of the carboxy-terminal truncations of yeast SmB; black and red arrowheads denote viable and lethal truncations, respectively. (C) The wild-type and truncated SMB1 alleles were tested for activity by plasmid shuffle in smb1▵ mud2▵ and smb1▵ mud1▵ strains. The growth phenotypes of viable FOA-resistant smb1▵ strains bearing the indicated SMB1 alleles are shown. Synthetic defects are denoted by •.

FIGURE 6.

Genetic interactions of SmB-Ala mutants. Yeast smb1▵ strains bearing the indicated SMB1-Ala allele on a CEN HIS3 plasmid in an otherwise wild-type (top panel), mud2▵, mud1▵, nam8▵, or tgs1▵ background as indicated were spot-tested for growth on YPD agar at the temperatures specified. Synthetic growth defects are denoted by •.

FIGURE 7.

Effects of simultaneous Sm site mutations in SmD3 and SmB. SMD3 (WT) and SMD3-Ala alleles on CEN LEU2 plasmids were cotransformed with SMB1 (WT) and SMB1-Ala alleles on CEN HIS3 plasmids into a yeast smd3▵ smb1▵ p(CEN URA3 SMD3 SMB1) strain. The indicated viable FOA-resistant strains recovered after plasmid shuffle were spot tested for growth on YPD agar at the temperatures specified. SMD3 SMB1 allele pairs listed at the bottom failed to complement smd3▵ smb1▵ in the plasmid shuffle assay and were deemed synthetically lethal.

FIGURE 8.

Genetic interactions of Snp1 amino-terminal truncations. (Top) Alignment of the amino-terminal amino acid sequences of the S. cerevisiae Snp1 and human U1-70K. Positions of side chain identity/similarity are indicated by • above the alignment. Arrowheads indicate the boundaries of the ▵N truncations of Snp1. (Bottom) Yeast snp1▵ strains bearing the indicated SNP1-▵N allele on a CEN HIS3 plasmid in an otherwise wild-type (top panel), mud2▵, mud1▵, nam8▵, or tgs1▵ background as indicated were spot-tested for growth on YPD agar at the temperatures specified. Synthetic growth defects are denoted by •.

FIGURE 9.

Genetic interactions of SmD3 with U2 snRNP subunit Lea1. Yeast lea1▵ strains bearing the indicated SMD3 alleles on a CEN LEU2 plasmid were spot-tested for growth on YPD agar at the temperatures specified.