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

Abstract

A seven-subunit Lsm2-8 protein ring assembles on the U-rich 3' end of the U6 snRNA. A structure-guided mutational analysis of the Saccharomyces cerevisiae Lsm2-8 ring affords new insights to structure-function relations and genetic interactions of the Lsm subunits. Alanine scanning of 39 amino acids comprising the RNA-binding sites or intersubunit interfaces of Lsm2, Lsm3, Lsm4, Lsm5, and Lsm8 identified only one instance of lethality (Lsm3-R69A) and one severe growth defect (Lsm2-R63A), both involving amino acids that bind the 3'-terminal UUU trinucleotide. All other Ala mutations were benign with respect to vegetative growth. Tests of 235 pairwise combinations of benign Lsm mutants identified six instances of inter-Lsm synthetic lethality and 45 cases of nonlethal synthetic growth defects. Thus, Lsm2-8 ring function is buffered by a network of internal genetic redundancies. A salient finding was that otherwise lethal single-gene deletions lsm2Δ, lsm3Δ, lsm4Δ, lsm5, and lsm8Δ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad lsmΔ lsmΔ double-deletions and lsmΔ lsmΔ lsmΔ triple-deletions. We find that U6 overexpression also rescues a lethal deletion of the U6 snRNP protein subunit Prp24 and that Prp24 overexpression bypasses the essentiality of the U6-associated Lsm subunits. Our results indicate that abetting U6 snRNA is the only essential function of the yeast Lsm2-8 proteins.

Keywords: Lsm proteins; Prp24; U6 snRNA; mRNA splicing.

FIGURE 1.

Topology and RNA interface of the Lsm2–8 ring. (A) Structure of the yeast Lsm2–8 ring in complex with 5′-GUUUU RNA (Zhou et al. 2014a, pdb 4M7A). The Lsm subunits are shown as cartoon traces and color-coded as indicated. The RNA is depicted as a stick model. (B) Stereo view (wall-eyed) of the RNA interface of the Lsm2 and Lsm3 subunits, which contact the 3′-terminal UpUpU trinucleotide. The base-stacking and hydrogen-bonding interactions of the RNA-binding triad amino acids (Phe35, Asn37, and Arg63 in Lsm2; His36, Asn38, and Arg69 in Lsm3) are shown. The structural images were prepared in Pymol.

FIGURE 2.

Structure guided mutagenesis of the five essential U6-associated Lsm subunits. (A) The primary structures of S. cerevisiae Lsm2, Lsm3, Lsm4, Lsm5, and Lsm8 are shown. The secondary structure elements (from the structure in Fig. 1A) are depicted above the amino acid sequences as magenta β strands (arrows) and cyan α helices (cylinders). The amino acids subjected to alanine scanning mutagenesis are shaded in gold (RNA-binding triads), green (conserved Asp that stabilizes the β2–β3 RNA-binding loop), or blue (intersubunit interface). The C terminus of the Lsm4-CΔ92 truncation mutant is indicated by the reverse arrowhead. (B) The wild-type and mutated LSM alleles were tested for lsmΔ complementation by plasmid shuffle. Lethal alleles that failed to support growth on FOA are highlighted in red boxes. Viable LSM-Ala and LSM4-CΔ92 strains were spot-tested for growth on YPD agar at 18°C–37°C. Those that grew as well as wild-type LSM cells (+++) are highlighted in gray boxes. Strains that were very sick (tiny colonies at all temperatures) are highlighted in yellow boxes.

FIGURE 3.

Mutational synergies with lsm2Δ. (A–D) Synthetically lethal pairs of alleles are highlighted in red boxes. Other negative pairwise interactions are classified as sick or very sick (yellow boxes), or temperature-sensitive (ts) or cold-sensitive (cs) (light green boxes). Gray boxes denote lack of mutational synergy.

FIGURE 4.

Intersubunit synthetic genetic interactions. (A–F) Synthetically lethal pairs of alleles are highlighted in red boxes. Other negative pairwise interactions are classified as sick or very sick (yellow boxes), or temperature-sensitive (ts) or cold sensitive (cs) (light green boxes). Gray boxes denote lack of mutational synergy.

FIGURE 5.

Bypass of essential Lsm subunits by U6 snRNA overexpression. The indicated lsmΔ strains were tested by plasmid shuffle for complementation by the corresponding wild-type LSM genes (as positive controls) and for bypass by a 2µ-U6 plasmid. The viable FOA-resistant lsmΔ p[CEN LSM] and lsmΔ p[2µ-U6] strains were spot-tested for growth on YPD agar at the temperatures specified.

FIGURE 6.

Bypass of lsm double and triple deletions by U6 snRNA overexpression. (A) The indicated lsmΔ lsmΔ strains were tested by plasmid shuffle for complementation by the corresponding pairs of wild-type LSM genes (as positive controls) and for bypass by a 2µ-U6 plasmid. The viable FOA-resistant controls and the lsmΔ lsmΔ p[2µ-U6] strains were spot-tested for growth on YPD agar at the temperatures specified. (B) The indicated triple-deletion strains harboring a 2µ-U6 plasmid were spot-tested for growth on YPD agar in parallel with a wild-type strain.

FIGURE 7.

Bypass of prp24Δ by U6 overexpression and bypass of lsmΔ by Prp24 overexpression. (A) prp24Δ p[URA3 CEN PRP24] cells were tested by plasmid shuffle for complementation by a 2µ PRP24 plasmid (as positive control) and for bypass by a 2µ-U6 plasmid. (B) The indicated lsmΔ and lsmΔ lsmΔ strains were tested by plasmid shuffle for bypass by a 2µ-PRP24 plasmid. The viable FOA-resistant strains were spot-tested for growth on YPD agar at the temperatures specified.

FIGURE 8.

Overexpression of Prp24 suppresses lethality of usb1Δ. usb1Δ p[URA3 CEN USB1] cells were tested by plasmid shuffle for complementation by a CEN USB1 plasmid (as positive control) and for bypass by 2µ-U6 and 2µ-PRP24 plasmid. The viable FOA-resistant strains were spot-tested for growth on YPD agar at the temperatures specified.