Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena

Abstract

RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.

FIGURE 1:

Amino acid sequence alignment of T. thermophila Rnt2 family members. Absolutely conserved residues are indicated with black background, and residues conserved in five to eight sequences are indicated with gray background. The two active-site motifs CAS I and CAS II are indicated below the sequence alignment, and the two catalytic histidines are marked with an asterisk above. The conserved cysteines involved in disulfide bridges are also indicated above the sequence alignment and are numbered according to previous annotation (Irie, 1999). The N-terminal signal peptides predicted by SignalP are underlined.

FIGURE 2:

Cellular phenotypes associated with Rnt2 depletion. DAPI staining of macronuclei and micronuclei in wild-type CU522 compared with Rnt2 A KD or Rnt2 B KO cells with BigMac or MultiMic pheno­types, respectively (arrows). Scale bars, 20 μm.

FIGURE 3:

Profiles of tRNA fragments in Rnt2 KO strains. (A) Southern blot analysis confirming complete replacement of macronuclear RNT2 genes with the resistance cassette neo2 or bsr2. Genomic DNA was digested with the indicated restriction enzymes to resolve wild-type (wt) from KO chromosomes as schematized above each blot. Two or three independent clonal cell lines are compared with wt for each RNT2 locus targeting. (B) Total RNA from control and KO strains grown in defined complete synthetic medium without or with transfer to starvation medium for a subsequent 3 h. RNA was analyzed by SYBR Gold stain (top) and by Northern blot hybridization with a probe against the 3′ end of tRNA Gly (bottom; only tRNA-relevant blot sections are shown). The positions of mature tRNA and tRNA halves are indicated. An arrow highlights the Rnt2 C KO lane with an altered size of tRNA halves. (C) RNA from cells grown in complex, rich medium NEFF with or without subsequent transfer to starvation medium. RNA was analyzed by Northern blot hybridization with probes against tRNA Gly 5′ and 3′ ends.

FIGURE 4:

Elevated levels of tRNA and rRNA fragments in Rnt2 double-KO strains. (A) Southern blot analysis confirming complete replacement of a second RNT2 locus with the bsr2 resistance cassette. Genomic DNA from wild-type (wt) cells and three independent double-targeted clonal cell lines was digested with appropriate restriction enzymes to resolve wild-type and KO chromosomes as schematized above each blot. (B) RNA harvested from growing cells or cells starved for 3.5 h from two independent cell lines for each Rnt2 double KO, stained with SYBR Gold (left). Results are shown from Northern blot hybridization to detect 5′ and 3′ fragments of tRNA Gly and tRNA Ala (right). (C) Northern blot hybridization analysis of RNA as earlier described. Results from probes complementary to 5S rRNA, 5.8S rRNA, SRP RNA, small nuclear RNA U6, and small nucleolar RNATtnuCD32 are shown.

FIGURE 5:

Accumulation levels of tRNA fragments compared across culture conditions. The accumulation of 5′ (top) and 3′ (bottom) tRNA Gly fragments upon various stresses: starvation, stationary phase, mild or severe cold shock (CS), and mild or severe heat shock (HS). The positions of mature tRNAs, tRNA halves, and tRNA quarters, as well as RNA size markers, are indicated.

FIGURE 6:

Evidence for an RNP-free state of tRNA halves in cell extract. RNA was harvested from every second fraction of a glycerol gradient fractioning cell extract from cold-shocked cells. SYBR Gold–stained RNA (top) and Northern blot hybridization of the same RNA transferred to a membrane (bottom) probed with an oligonucleotide complementary to the tRNA Gly 3′ end are shown. Size markers and tRNA species are indicated.