Starvation-induced Cleavage of the tRNA Anticodon Loop in Tetrahymena thermophila

Suzanne R Lee,Kathleen Collins

doi:10.1074/jbc.m510356200

Suzanne R Lee, Kathleen Collins

Open Access

https://doi.org/10.1074/jbc.m510356200

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Abstract

Amino acid deprivation triggers dramatic physiological responses in all organisms, altering both the synthesis and destruction of RNA and protein. Here we describe, using the ciliate Tetrahymena thermophila, a previously unidentified response to amino acid deprivation in which mature transfer RNA (tRNA) is cleaved in the anticodon loop. We observed that anticodon loop cleavage affects a small fraction of most or all tRNA sequences. Accumulation of cleaved tRNA is temporally coordinated with the morphological and metabolic changes of adaptation to starvation. The starvation-induced endonucleolytic cleavage activity targets tRNAs that have undergone maturation by 5' and 3' end processing and base modification. Curiously, the majority of cleaved tRNAs lack the 3' terminal CCA nucleotides required for aminoacylation. Starvation-induced tRNA cleavage is inhibited in the presence of essential amino acids, independent of the persistence of other starvation-induced responses. Our findings suggest that anticodon loop cleavage may reduce the accumulation of uncharged tRNAs as part of a specific response induced by amino acid starvation.

Highlights

Transfer RNA serves a second, less well characterized role in gene expression by acting as a signaling molecule
Starvation Induces Cleavage of transfer RNA (tRNA) in the Anticodon Loop—In characterizing the small noncoding RNAs expressed in starving T. thermophila, we observed a population of ϳ30 –35-nucleotide RNAs on gels stained directly for RNA with SYBR Gold (Fig. 1)
Starvation is uncoupled from initiation in 50 mM Tris, which induces a starvation response but blocks cells before initiation is complete. Under this condition of starvation, the degradation of rRNA and proteins still occurs [18], and G8 RNA was induced (Fig. 5A, lane 8, bottom), but tRNA cleavage was prevented (Fig. 5A, lane 8, top). These results suggest that the induction of anticodon loop cleavage of tRNAs is coordinated with life cycle progression, rather than occurring solely in response to the absence of essential amino acids

Summary

EXPERIMENTAL PROCEDURES

Strains and Cell Culture—For vegetative growth and starvation, T. thermophila strain SB210 was cultured as described [19]. RNA Detection, Cloning, and Identification—Total RNA was isolated from whole cells using Trizol (Invitrogen). To detect tRNAs by Northern blotting, RNA was transferred to Hybond Nϩ membranes (Amersham Biosciences) and probed with 5Ј. End-labeled DNA oligonucleotides corresponding to the terminal 22 nucleotides, excluding the 3Ј CCA, of individual tRNAs. As a control for induction of starvation, Northern blot probes were designed against the noncoding RNA G8 [23, 24]. DNA/RNA adapters (Integrated DNA Technologies, see below) with BanI sites were ligated to sRNA ends with T4 RNA ligase (Amersham Biosciences). Concatamers of BanI-digested RT-PCR products obtained with T4 DNA ligase (200 units/ml, New England Biolabs) were treated with alkaline phosphatase and TOPO-cloned (Invitrogen).

RESULTS

TABLE ONE

DISCUSSION