tRNA cleavage is a conserved response to oxidative stress in eukaryotes

Abstract

Recent results have identified a diversity of small RNAs in a wide range of organisms. In this work, we demonstrate that Saccharomyces cerevisiae contains a small RNA population consisting primarily of tRNA halves and rRNA fragments. Both 5' and 3' fragments of tRNAs are detectable by Northern blot analysis, suggesting a process of endonucleolytic cleavage. tRNA and rRNA fragment production in yeast is most pronounced during oxidative stress conditions, especially during entry into stationary phase. Similar tRNA fragments are also observed in human cell lines and in plants during oxidative stress. These results demonstrate that tRNA cleavage is a conserved aspect of the response to oxidative stress.

FIGURE 1.

The Saccharomyces cerevisiae small RNA population includes tRNA pieces. (A) Total yeast RNA (Y) 3′-end-labeled with [³²P]-pCp separated on a 20% polyacrylamide gel. The small RNA population is indicated. (B) 5′- and 3′-specific tRNA probes identify discrete bands on Northern blots. tRNA glyphs indicate full-length and fragment species. (C) Estimated cleavage sites for tRNAs shown in B are indicated. (D) tRNA fragmentation does not increase in strains defective for exonucleolytic decay. The indicated strains and WT control were grown to mid-log phase and harvested for RNA analysis. 5′-tRNA^His _GTG probe. (L) ϕX174/Hinf 1 ladder. Ladder sizes are indicated in nucleotides. All experiments were repeated at least three times; representative blots are shown.

FIGURE 2.

rRNA cleavage increases in response to oxidative stress. (A) Location of rRNA fragments from the small-RNA library mapped onto the 35s rRNA sequence, with component 5.8s, 18s, and 25s rRNAs indicated. (B) rRNA fragments increase in response to oxidative stress from H₂O₂ exposure. 3′ 25s rRNA probe. (C) rRNA fragments increase during entry into stationary phase; samples collected over 6 d are compared to a sample from cells in mid-log phase (“C”), to highlight the increase in cleavage. 3′ 25s rRNA probe. Arrow indicates the 3′ rRNA fragment discussed in the text. Ladder sizes are indicated in nucleotides. All experiments were repeated at least three times; representative blots are shown.

FIGURE 3.

tRNA cleavage is not a quality control process. (A) The cca1-1 strain after growth at the nonpermissive temperature (37°C) for the indicated times. 5′-tRNA^His _GTG probe. (B) hts1.1 cells and control cells grown at the nonpermissive temperature (39°C) for the indicated times. yRP841 is the WT control strain. 5′-tRNA^His _GTG probe. (C) trm8Δ, trm82Δ cells and WT control, grown in glycerol at 37°C for 4 h. 3′-tRNA^Met _CAT probe. tRNA glyphs indicate full-length and fragment species. (L) ϕX174/Hinf 1 ladder. Ladder sizes are indicated in nucleotides. Experiments was repeated at least three times; representative blots are shown.

FIGURE 4.

Stress conditions induce tRNA cleavage in yeast. (A) Heat stress leads to an increase in tRNA fragment level. WT yeast were harvested at the indicated times after growth at 30°C or 37°C. 3′-tRNA^Lys _CTT probe. (B) tRNA fragment production during amino acid starvation. Cells were collected after 2 h of starvation for the indicated amino acid. 3′-tRNA^Asp _GTC probe. (C) Stationary phase entry coincides with a dramatic increase in tRNA fragment. Samples collected over 6 d are compared to a sample from cells in mid-log phase (“C”), to highlight the increase in cleavage. 3′-tRNA^Arg _TCT probe. (D) UV exposure does not significantly increase tRNA cleavage in yeast. Yeast treated with 0 or 50 J/m² UV were harvested at the indicated times. 3′-tRNA^Lys _CTT probe. (E) Glucose stress does not significantly increase tRNA fragment production in yeast. WT yeast were grown to mid-log in YEPD, then shifted to YEPD (lane 2), SC medium with glucose (lane 3), or SC medium without glucose (lane 4), and collected after 4 h. 3′-tRNA^Lys _CTT probe. (F) Nitrogen starvation increases tRNA fragment production. Yeast cells were grown in media with or without a nitrogen source for 4 h. 3′-tRNA^His _GTG probe. tRNA glyphs indicate full-length and fragment species. (L) ϕX174/Hinf 1 ladder. Ladder sizes are indicated in nucleotides. All experiments were repeated at least three times; representative blots are shown.

FIGURE 5.

Oxidative stress increases tRNA fragment production in yeast, Arabidopsis, and mammalian cells. (A) WT yeast were exposed to 0 or 0.4 mM H₂O₂ for 200 min. 3′-tRNA^Met _CAT probe. (B) Two-week-old Arabidopsis seedlings were treated with 0 or 10 mM H₂O₂ for 2 or 4 h. 5′-tRNA^His _GTG probe. (C) Subconfluent human HeLa cells were treated with 0 or 5 mM H₂O₂ for 4 h. 3′-tRNA^His _GTG probe. tRNA glyphs indicate full-length and fragment species. (L) ϕX174/Hinf 1 ladder. Ladder sizes are indicated in nucleotides. All experiments were repeated at least three times; representative blots are shown.

FIGURE 6.

Additional examples of tRNA cleavage in Arabidopsis. (A) tRNA cleavage shows a tissue-specific pattern in plants, and accumulates at a higher level in flowers (F) than in seedlings (S). 5′-tRNA^GLU _CTC probe. (B) H₂O₂-induced cleavage differs among tRNAs. Two-week-old seedlings treated with 0 (C) or 10 mM H₂O₂ for the indicated times were analyzed using the indicated probes. tRNA glyphs indicate full-length and fragment species.

FIGURE 7.

tRNA cleavage in mammalian cells is not a general response to all apoptotic inducers. (A) HeLa cells were treated with 5 mM H₂O₂ or mock-treated for the indicated times. Arrow indicates when cells were first observed rounding up and detaching. 3′-tRNA^His _GTG probe. (B–D) ARPE-19 cells at subconfluence were used for these experiments. In B, cells were exposed to the apoptotic inducer α-FAS (1 μg/mL) or mock-stressed for 72 h. 3′-tRNA^His _GTG probe. In C, cells were exposed to the apoptotic inducer staurosporine in DMSO (1 μM) or DMSO alone for 4 h. 3′-tRNA^His _GTG probe. For D, cells were treated with 16 mM caffeine for the indicated times. 3′-tRNA^His _GTG probe. tRNA glyphs indicate full-length and fragment species. (L) ϕX174/Hinf 1 ladder. Ladder sizes are indicated in nucleotides. All experiments were repeated at least three times; representative blots are shown.