3' poly(A) is dispensable for translation

Abstract

In wild-type cells, the 3' poly(A) structure is necessary for translation of mRNA and for mRNA stability. The superkiller 2 (ski2), ski3, ski6, ski7, and ski8 mutations enhance the expression of the poly(A)(-) mRNAs of yeast RNA viruses. Ski2p is a DEVH-box RNA helicase and Slh1p resembles Ski2p. Both repress L-A double-stranded RNA (dsRNA) virus copy number, further suggesting that their functions may overlap. We find that slh1Delta ski2Delta double mutants are healthy (in the absence of viruses) and show normal rates of turnover of several cellular mRNAs. The slh1Delta ski2Delta strains translate electroporated nonpoly(A) mRNA with the same kinetics as polyA(+) mRNA. Thus, the translation apparatus is inherently capable of efficiently using nonpoly(A) mRNA even in the presence of normal amounts of competing poly(A)(+) mRNA, but is normally prevented from doing so by the combined action of the nonessential proteins Ski2p and Slh1p.

Figure 1

Normal growth at 30°C of ski2Δ slh1Δ cells. Isogenic wild type, ski2Δ, slh1Δ, and ski2Δ slh1Δ mutants were grown on yeast extract/peptone/dextrose plates at 30°C for 3 days.

Figure 2

Elevated L-A and L-BC viral dsRNA copy number in slh1 ski2 strains. Two colonies of each strain were grown. Total RNA (5 μg) was loaded onto a 1.2% formaldehyde-agarose gel. The size-fractionated RNAs were transferred to Hybond-N+ membranes (Amersham International) and hybridized under high-stringency conditions to radiolabeled RNA probes corresponding to 18S ribosomal RNA and the ds L-A or L-BC RNAs. After hybridization, the membranes were exposed to film for 16 h unless otherwise noted.

Figure 3

ski2Δ slh1Δ strains translate A+ and A− mRNAs alike. Isogenic strains 3221 (wild type), 3515 (ski2Δ), 4106 (slh1Δ), and 4107 (ski2Δslh1Δ) were electroporated (1.3 × 10⁸ cells) with 2 μg of RNA. Cells were maintained at 25°C and assayed for luciferase activity at the indicated timepoints as described (9). Protein concentrations of all lysates were measured by the Bio-Rad protein assay kit. The functional half-lives of each mRNA were calculated by determining the time required for each to produce 50% maximal luciferase activity (1). The maximum rate of luciferase synthesis was the maximum rate change of the activity curves (generally during the first 20–40 min of the assay). The results shown are an average of three experiments. The maximum luciferase translation rate (<0.4 units/μg cell protein/min) is about 10⁻⁶ part of total protein synthesis in these cells. (□) C+ A+; (■) C+ A−; (●) C− A+; and (○) C− A−.

Figure 4

ski2Δ ski6–2 strains translate A+ and A− mRNAs alike. Strains 3221 (wild type) and 4709 (ski2Δ ski6–2) were electroporated with 2 μg of luciferase mRNA and assayed for luciferase production as in Fig. 2.