Jinmi Kim

The DEAD-box RNA helicase, Dhh1, functions in mating by regulating Ste12 translation in Saccharomyces cerevisiae.

The DEAD-box RNA helicase, Dhh1, is a member of a highly conserved subfamily designated RCK/p54 helicases. Dhh1 functions as mRNA decapping activator, and is localized to discrete cytoplasmic foci known as processing bodies (P-bodies). Here, we describe the essential roles of Dhh1 in the yeast mating pathway. A dhh1 deletion mutation caused a significant decrease in the protein level of Ste12, a mating-specific transcription factor, resulting in severe mating defects. We examined the accumulation of Dhh1-GFP in P-bodies during mating. Following pheromone treatment, the P-body intensity and number increased in wild-type cells, while dhh1 mutant cells failed to show P-body formation. Both the mating and P-body phenotypes of dhh1 were suppressed by overexpression of STE12 or CAF20 encoding an eIF4E inhibitor. In wild-type cells, CAF20 overexpression led to an increased level of Ste12 protein as well as highly developed P-bodies. We propose that Dhh1 and Caf20 regulate the Ste12 protein expression and the Ste12 protein level is associated with P-body formation during mating.

Upstream open reading frames regulate the cell cycle-dependent expression of the RNA helicase Rok1 in Saccharomyces cerevisiae

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.

Proteomic analysis of hyphae-specific proteins that are expressed differentially in cakem1/cakem1 mutant strains of Candida albicans

The yeast-to-hyphal transition is a major virulence factor in the fungal pathogen Candida albicans. Mutations in the CaKEM1 gene, which encodes a 5′-3′ exoribonuclease responsible for mRNA degradation, show a defect in hyphal growth. We applied two-dimensional gel electrophoresis to identify hyphae-specific proteins that have altered expressions in the presence of the cakem1 mutation. Eight proteins, Eno1, Eps1, Fba1, Imh3, Lpd1, Met6, Pdc11, and Tsa1 were upregulated during hyphal transition in wild-type but not in cakem1/cakem1 mutant cells. A second group of proteins, Idh1, Idh2, and Ssb1, showed increased levels of expression in cakem1/cakem1 mutant cells when compared to wild-type cells. Overexpression of Lpd1, a component of the pyruvate dehydrogenase complex, caused slight hyperfilamentation in a wild-type strain and suppressed the filamentation defect of the cakem1 mutation. The Ssb1 protein, which is a potential heat shock protein, and the Imh3 protein, which is a putative enzyme in GMP biosynthesis also showed the filamentation-associated phenotypes.

Deletion analysis of LSm, FDF, and YjeF domains of Candida albicans Edc3 in hyphal growth and oxidative-stress response

Candida albicans is an opportunistic fungal pathogen whose responses to environmental changes are associated with the virulence attributes. Edc3 is known to be an enhancer of the mRNA decapping reactions and a scaffold protein of cytoplasmic processing bodies (P-bodies). Recent studies of C. albicans Edc3 suggested its critical roles in filamentous growth and stress-induced apoptotic cell death. The edc3/edc3 deletion mutant strain showed increased cell survival and less ROS accumulation upon treatment with hydrogen peroxide. To investigate the diverse involvement of Edc3 in the cellular processes, deletion mutations of LSm, FDF, or YjeF domain of Edc3 were constructed. The edc3-LSmΔ or edc3-YjeFΔ mutation showed the filamentation defect, resistance to oxidative stress, and decreased ROS accumulation. In contrast, the edc3-FDFΔ mutation exhibited a wild-type level of filamentous growth and a mild defect in ROS accumulation. These results suggest that Lsm and YjeF domains of Edc3 are critical in hyphal growth and oxidative stress response.

Mutational analysis of the RNA helicase Dhh1 in Ste12 expression and yeast mating

Dhh1 and Dhh1 homologues (RCK/p54/DDX6) are members of the DEAD-box protein family of RNA helicases. These proteins display conserved sequence motifs for ATPase and RNA binding activities. Dhh1 is a component of the P-bodies (processing bodies) of mRNA granules and functions as an mRNA decapping activator in Saccharomyces cerevisiae. Dhh1 also contributes to gene-specific regulation during yeast mating. The dhh1 deletion mutation results in a significant decrease in the expression of Ste12, a mating-specific transcription factor, showing severe mating defects. Here, we introduced amino-acid substitution mutations in the ATPase and RNA binding domains of Dhh1 and also constructed a deletion of 79 amino acids at the Q/P-rich C-terminal region. The mutations in ATPase A and B motif (K96R, D195A) and C-terminus deletion showed reduced levels of mating efficiency as well as Ste12 protein expression. The Q/P-rich C-terminal region of Dhh1 was dispensable for growth at nonpermissive temperature 37°C but appeared to play an important role in regulating the Ste12 protein expression and mating processes. The P-body accumulation induced by treatment with α-mating factor required ATPase, RNA-binding and the Q/P-rich C-terminal domains of Dhh1.

Identification of Psk2, Skp1, and Tub4 as trans-acting factors for uORF-containing ROK1 mRNA in Saccharomyces cerevisiae.

Rok1, a DEAD-box RNA helicase, is involved in rRNA processing and the control of cell cycle progression in Saccharomyces cerevisiae. Rok1 protein expression is cell cycle-regulated, declining at G1/S and increasing at G2. The downregulation of Rok1 expression in G1/S phase is mediated by the inhibitory action of two upstream open reading frames (uORFs) in the ROK1 5′-untranslated region (5′UTR). We identified Psk2 (PAS kinase), Skp1 (kinetochore protein) and Tub4 (γ-tubulin protein) as ROK1 5′UTR-interacting proteins using yeast three-hybrid system. A deletion analysis of PSK2 or inactivation of temperature-sensitive alleles of SKP1 and TUB4 revealed that Rok1 protein synthesis is repressed by Psk2 and Skp1. This repression appeared to be mediated through the ROK1 uORF1. In contrast, Tub4 plays a positive role in regulating Rok1 protein synthesis and likely after the uORF1-mediated inhibitory regulation. These results suggest that 5′UTR-interacting proteins, identified using three hybrid screening, are important for uORF-mediated regulation of Rok1 protein expression.

Roles of eIF4E-binding protein Caf20 in Ste12 translation and P-body formation in yeast

Translation initiation factor eIF4E forms eIF4E-eIF4G complex at the 5’ cap of mRNA. This interaction can be inhibited by the family of 4E-binding proteins (4E-BP). In yeast Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete with eIF4G for binding to eIF4E via the shared conserved interaction motif. In order to investigate the roles of Caf20 in gene-specific translational regulation and the formation of mRNA granules (P-bodies), we introduced substitution mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding motif for CAF20. Overexpression of the wild-type CAF20 showed an increased protein level of Ste12 transcription factor as well as highly developed P-body formation. However, 4E-binding site mutations of CAF20 led to a reduced number of P-body foci and decreased levels of Ste12 protein. The phenotypes of the caf20 deletion mutation were also analyzed, and we suggest that Caf20 plays a critical role in Ste12 protein expression and in the control of P-body formation.