Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes?

Abstract

The T(2) family of nonspecific endoribonucleases (EC ) is a widespread family of RNases found in every organism examined thus far. Most T(2) enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA. Although the biological functions of various T(2) RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown. In the present work, we characterized Rny1, the only T(2) RNase in Saccharomyces cerevisiae. Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress. Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth. These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases. Additionally, the complementation depends on RNase activity. When coupled with a recent report on the effect of specific RNAs on membrane permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. & Yarus, M. (1999) Proc. Natl. Acad. Sci. USA 96, 10649-10654], our work suggests an unexpected role for Rny1 and possibly other secretory RNases. These enzymes may regulate membrane permeability or stability, a hypothesis that could present an alternative perspective for understanding their functions.

Figure 1

RNY1 characterization. (A) Predicted Rny1 amino acid sequence reveals the presence of the two conserved sequences characteristic of the RNase T₂ family of RNases (black boxes), four N-glycosylation sites (asterisk), a secretion signal (dotted line), and a C-terminal extension (gray box) containing two putative nuclear localization signals (diagonal bars). (B) RNY1 deletion strategy. Haploid yeast cells carrying a disrupted rny1 gene were produced by homologous recombination after introducing into WT cells a Hindlll–Dral fragment from plasmid p1711 that contains the selectable marker URA3 replacing part of the RNY1 ORF between the two Sful sites.

Figure 2

Rny1 is an active RNase and is glycosylated highly. (A) Secreted proteins recovered from the growth media (12 μg per lane) or intracellular proteins (40 μg per lane) were analyzed by using a gel-based RNase assay. Proteins were prepared from WT and rny1Δ cells and from WT and rny1Δ cells transformed with the plasmid p2023 that express the RNY1 gene constitutively. The asterisk indicates the main intracellular activity (≈70 kDa). (B) Secreted proteins from an RNY1 overexpressing cell culture (10 μg) were incubated at 37°C for the indicated period of time with or without the addition of 4 units of PNGase F and then analyzed by using a gel-based RNase assay or by SDS/PAGE and Coomassie blue staining.

Figure 3

(A) The RNY1 promoter contains several putative stress-regulated elements. Arrows indicate the position of the different binding elements. HSF, heat shock factor; AP-1, yeast AP-1; ROX1, Rox1-binding site; STRE, stress response element. The numbers indicate the distance from the ATG codon (+1). (B) RNY1 expression is regulated by different stress conditions. WT cells were grown to early exponential phase at 25°C and diluted to an optical density at 600 nm of 0.1. Half the culture was incubated at 25°C, and the other half was shifted to 37°C or kept at 25°C but with the addition of NaCl to a final concentration of 0.3 M. Aliquots (2 ml) were harvested at the indicated time points and total RNA was analyzed with Northern blot by using RNY1 or ACT1 probes.

Figure 4

Phenotype of rny1Δ strain in the PSY316 background. (A) Cells (PSY316 background) were grown in YPD plates or YPD plus 50 g/liter NaCl plates at 25° or 37°C, as indicated. For complementation experiments, rny1Δ cells were transformed with plasmid containing RNY1, the A. thaliana RNS1 gene, the mammalian gene for pancreatic RNase A, or empty vector (pG1 or YEpWL). (B) WT or rny1Δ cells (PSY316 background) were grown to exponential phase in SD medium at 25°C. Cells were viewed by using differential interference contrast microscopy. v, vacuole. (Bar = 5 μm.) (C) rny1Δ cells were transformed with plasmid containing the mammalian gene for pancreatic RNase A (WT) or the RNase A mutant H119D(H119D) and grown in SD plates at 25° or 37°C, as indicated.