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 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 deletionsΔ, Δ,Δ, , andΔ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad ΔΔ double-deletions and ΔΔ Δ 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.

Yeast U6 snRNA made by RNA polymerase II is less stable but functional

“…To test if this occurs, we first looked for genetic interactions between U6-II-Sm and Lsm proteins. Single-gene deletions of lsm5 and lsm8 are not lethal if U6 snRNA is over-expressed, consistent with the primary function of the Lsm2-8 proteins in yeast being stabilization of U6 snRNA (Pannone et al 2001; Roth et al 2018). We predicted that if Lsm5 or Lsm8 association with U6-II-Sm is not essential, then U6-II-Sm yeast cells should be viable in lsm511 or lsm811 strains after selection for loss of complementing URA3 -marked plasmids.…”

“…It should be noted that lsm5 and lsm8 deletions were viable in the presence of WT U6 ( Fig. S4 ), likely due to amplification of the low-copy plasmid containing the U6 gene (Pannone et al 2001; Roth et al 2018). The stronger requirement for Lsm5 and Lsm8 observed with U6-II-Sm may have to do with its decreased function, noted above.…”

Comparative transcriptome and proteome reveal synergistic functions of differentially expressed genes and proteins implicated in an over‐dominant silkworm heterosis of increased silk yield

“…Lsm3 is a member of Sm‐like protein family participating in pre‐mRNA splicing and decay (Roth et al, 2018). Lsm proteins consist of 8 members, and they form a cytoplasmic complex Lsm1‐7 participating in mRNA decay by promoting the decapping of target mRNAs, and a nuclear complex Lsm2‐8 participates in RNA splicing by binding to U6 small nuclear ribonucleoprotein (snRNA), a component of spliceosome.…”

Determining the Prognostic Value of Spliceosome-Related Genes in Hepatocellular Carcinoma Patients

“…The spliceosome is an organelle-like complex that plays a significant role in regulating gene expression and producing protein diversity ( Nilsen and Graveley, 2010 ). The spliceosome consists of five small nuclear ribonucleoproteins (snRNP; U1, U2, U4, U5, and U6), and the Lsm/sm proteins form the core scaffold of snRNP ( Roth et al, 2018 ). These molecules, including the snRNPs and Lsm proteins, are involved in the splicing of pre-mRNA.…”