C21orf57 is a human homologue of bacterial YbeY proteins

Abstract

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

Keywords: C21orf57; RNase; YBEY; Yeast.

Fig 1. Characterization of human YbeY, C21orf57 (HuYbeY)

(A) Protein sequence alignment of EcYbeY and HuYbeY. α-helices (α) and β-sheets (β) are indicated based on the crystal structure analysis of EcYbeY [10]. (B) PyMOL presentation of EcYbeY (PDB: 1XM5;[10]) showing the catalytic domain (left) and ribosome-binding domain (right). HuYbeY was modelled onto the coordinates of EcYbeY using SWISS-MODEL [6]. EcYbeY residues R59 (magenta), D85 (green) and H114 (blue) correspond to HuYbeY residues R55, D90 and H118, respectively, in (A) and (B). (C)-(F) MBP-HuYbeY degrades total human RNA. (C) RNase activity of MBP-HuYbeY protein in comparison to the mutated forms of MBP-HuYbeY protein; 1 μM concentration of protein was used. RNA was digested with 1 μM (D), 2.5 and 5 μM (E) concentration of MBP-HuYbeY protein, while the time-course assay was performed with 5 μM concentration of MBP-HuYbeY protein (F). Wild-type and mutant MBP-HuYbeY proteins in (C) have been purified from E. coli BL21(DE3) plysS Δrna strain; MBP-HuYbeY in (D, E and F) has been purified from E. coli BL21(DE3) plysS Δrna Δpnp strain. RNA digests were analyzed by agarose gel electrophoresis.

Fig 2. Expression of HuYbeY in E. coli MC4100 ΔybeY changes growth behavior and stress sensitivity

(A) Growth of ΔybeY cells carrying wild-type or mutant HuYbeY expression plasmids as indicated. Cells were grown in liquid medium at 37°C. (B) Cells were initially grown at 42°C and then shifted to 37°C. Averages of 9 transformants are shown in (A) and (B). None of the three mutants showed any activity. (C) Stress profile of ΔybeY cells carrying the respective HuYbeY expression plasmids grown on solid media at high temperature, after exposure to UV and in the presence of antibiotics cefotaxime and kasugamycin, respectively.

Fig 3. Expression of HuYbeY in yeast is toxic

S. cerevisiae CKY473 transformants expressing wild-type or mutant HuYbeY were grown on synthetic minimal medium containing 0.5% galactose supplemented with either 2% glucose, 2% raffinose or 3% glycerol as indicated. Plates were incubated at 30°C for 2 days (A), followed by a 7-day incubation at room temperature (B). Yeast cells carrying the empty vector are provided as control. Δmts indicates a HuYbeY expression construct lacking the predicted MTS. Assays were carried out in duplicates; a representative experiment is shown.