The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA Satoshi Kimuraa,b,c,1 and Matthew K. Waldora,b,c,1 aDivision of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA 02115; bDepartment of Microbiology, Harvard Medical School, Boston, MA 02115; and cHoward Hughes Medical Institute, Harvard Medical School, Boston, MA 02115 Edited by Susan Gottesman, NIH, Bethesda, MD, and approved December 11, 2018 (received for review August 15, 2018) The factors and mechanisms that govern tRNA stability in bacteria cations are often critical for efficient translation (9); consequently, are not well understood. Here, we investigated the influence of mutations that prevent such modification can result in loss of via- posttranscriptional modification of bacterial tRNAs (tRNA modifica- bility. In contrast, nucleotide modifications within the tRNA core tion) on tRNA stability. We focused on ThiI-generated 4-thiouridine [composed of the T- and D-stem loops and variable loop compo- (s4U), a modification found in bacterial and archaeal tRNAs. Compre- nents that interact within the tertiary structure (4)] and elbow hensive quantification of Vibrio cholerae tRNAs revealed that the [formed via the interaction of D and T loops (1)] are not generally abundance of some tRNAs is decreased in a ΔthiI strain in a station- required for viability, and the absence of a single modification ary phase-specific manner. Multiple mechanisms, including rapid usually does not have an effect on growth (15). Modifications out- degradation of a subset of hypomodified tRNAs, account for the side of the anticodon region are thought to promote the thermo- reduced abundance of tRNAs in the absence of thiI. Through trans- dynamic stability (i.e., structural rigidity) of tRNAs through poson insertion sequencing, we identified additional tRNA modifica- reinforcing the base interactions in the core or inhibiting unfavor- tions that promote tRNA stability and bacterial viability. Genetic able conformations (8). tRNAs’ highly stable structures are thought analysis of suppressor mutants as well as biochemical analyses to contribute to their intracellular stability; in general, tRNAs ex- revealed that rapid degradation of hypomodified tRNA is mediated hibit a high melting temperature in vitro and a long half-life within by the RNA degradosome. Elongation factor Tu seems to compete the cell. with the RNA degradosome, protecting aminoacyl tRNAs from decay. Although individual mutations in tRNA modification loci Together, our observations describe a previously unrecognized bac- do not generally have detrimental consequences, in yeast the terial tRNA quality control system in which hypomodification sensi- absence of multiple nonessential modifications results in a tizes tRNAs to decay mediated by the RNA degradosome. temperature-sensitive phenotype due to rapid exonuclease- mediated tRNA decay (RTD) of specific tRNA species (16– tRNA modification | RNA degradosome | thiI | Vibrio cholerae | 18). RTD and other mechanisms that degrade hypomodified or stationary phase mutated mature yeast tRNAs are thought to serve as a sur- veillance system to eliminate tRNA molecules that have in- ranslation of mRNAs into the proteins that they encode is correct nucleosides or conformations (19). Bacteria are not Tdependent on tRNAs that function as adapter molecules, known to have a similar surveillance system, and degradation of coupling the presence of specific mRNA codons to the incor- poration of corresponding amino acids into polypeptides. These Significance short noncoding RNA molecules fold into a highly conserved structure that is essential for effective charging by aminoacyl- The cellular mechanisms governing tRNA stability in bacteria tRNA synthetases, interaction with the ribosomal translation ma- are not well understood, and in contrast to eukaryotes, a chinery, and pairing with specific codons in mRNAs (1–3). bacterial system for degradation of mature tRNAs has not tRNAs adopt a “clover leaf” secondary structure that generally been described. We investigated the effect of thiolation on includes five characteristic elements: the accepter stem, the tRNA stability in Vibrio cholerae and found that this wide- D-stem loop (D arm), the anticodon stem loop (anticodon arm), spread posttranscriptional tRNA modification modulates the the variable loop, and the T-stem loop (T arm) (SI Appendix, Fig. stability of a subset of tRNAs in a stationary phase-specific S1A). tRNAs’ characteristic L-shaped tertiary structure results manner. Additional tRNA modifications were also found to from interactions between these elements (4). promote tRNA stability. Mechanistic analyses revealed that tRNAs are transcribed as longer precursor tRNA molecules, hypomodified tRNAs can be degraded by the RNA degrado- which then undergo endonucleolytic cleavage and exonucleolytic some, a multicomponent RNA degradation complex. Thus, the trimming of their 5′ and 3′ ends (5). Additional posttranscriptional degradosome serves as a previously unrecognized bacte- modifications are performed by a wide variety of dedicated tRNA- rial tRNA quality control system that mediates clearance of modifying enzymes (6), the activities of which include methylation, hypomodified tRNAs. pseudouridylation, sulfuration, and coupling of amino acids. Over 100 species of modifications have been discovered across all domains Author contributions: S.K. and M.K.W. designed research; S.K. performed research; S.K. contributed new reagents/analytic tools; S.K. analyzed data; and S.K. and M.K.W. wrote of life (7). Some modifications are introduced in nearly all tRNA the paper. species, whereas others are limited to a small subset of tRNA species. The authors declare no conflict of interest. Similarly, some modifications are present across all domains of life, This article is a PNAS Direct Submission. while others are limited to a single domain (8). tRNA-modifying enzymes have specificity both for particular tRNA species and for Published under the PNAS license. the sites to be modified within them (9). The extent of tRNA Data deposition: The data reported in this paper have been deposited in the NCBI Se- quence Read Archive, https://www.ncbi.nlm.nih.gov/sra (accession nos. SRP169512 and modification is not constant; instead, it can vary in response to SRP169513). cellular and environmental factors, such as growth rate and oxygen 1To whom correspondence may be addressed. Email: [email protected] or or nutrient levels (5, 10–14). [email protected]. Modifications are not uniformly distributed across the length of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tRNA molecules (8). Within the anticodon, the first or “wobble” 1073/pnas.1814130116/-/DCSupplemental. position is particularly subject to modification, and these modifi- Published online January 8, 2019. 1394–1403 | PNAS | January 22, 2019 | vol. 116 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1814130116 Downloaded by guest on September 27, 2021 hypomodified or thermodynamically destabilized mature tRNAs OD600 = 0.5 (late log phase) and 24 h after inoculation and then, has not been reported. However, mutated thermodynamically electrophoresed on polyacrylamide gels. No difference between unstable precursor tRNA is degraded by PNPase in Escherichia the bulk amounts of tRNAs present in the two strains was ob- coli (20). In addition, a temperature-sensitive decrease in the served (Fig. 1A). However, Northern blotting, using 53 probes abundance of some tRNAs was observed in a tRNA modification that detect all 55 tRNA species in V. cholerae (two probes detect mutant in Thermus thermophilus (21). Rapid decreases in the multiple tRNAs), revealed consistent thiI-dependent differences abundance of WT E. coli tRNA after stresses, such as amino acid in tRNA abundance, almost exclusively in stationary phase. We starvation and oxidative stress, were reported recently (22, 23), but found that 12 tRNA species were significantly less abundant in the pathways underlying these decreases have not been identified. stationary-phase samples from the ΔthiI strain (fc < 0.5 and P < In Vibrio cholerae, the cause of the diarrheal disease cholera, 0.05) (Fig. 1 A and B and SI Appendix, Fig. S2), with differences transposon insertion sequencing (TIS) analysis revealed that sev- reaching as high as 18-fold. tRNA-Tyr, tRNA-Ser1, tRNA-Cys1, eral tRNA modification loci are required for optimal bacterial and tRNA-Cys2 showed the most profound reductions (Fig. 1 A growth in an animal model of disease (24). These loci (thiI, miaA, and B). In log-phase samples, the abundance of only one tRNA mnmE,andmnmG) were not found to be required for V. cholerae (tRNA-Gln1A) was significantly reduced in the ΔthiI strain. These proliferation in nutrient-rich media, although the growth of mu- results suggest that ThiI and thus, the presence of s4U modifica- tants in culture tubes was not extensively characterized. Conse- tions modulate tRNA levels in a subset of tRNA species in a quently, mutations in these loci are thought to impair V. cholerae stationary phase-specific manner. growth in only a subset of conditions. With the exception of thiI, Almost all of the tRNA species with thiI-dependent reduced these genes encode enzymes that synthesize modifications within abundance in stationary phase are known to harbor s4UinE. coli the anticodon loop and thus, likely directly promote the efficiency (29), but the presence of this modification on V.