Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations

Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations Daan F. van den Berg, Baltus A. van der Steen, Ana Rita Costa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2166710/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract tRNAs in bacteriophage genomes are widespread across bacterial genera, but their exact function has remained unclear for more than 50 years. Multiple hypotheses have been proposed, with the most established being codon compensation, where codons more rarely used by the host but necessary for the phage are supplemented by tRNAs encoded by the phage. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs are a means to counteract the tRNA-depleting strategies of the host to defend from viral infection. Based on mutational patterns of tRNA anticodon loops, we predict that phage tRNAs are insensitive to the host tRNAses. For tRNAs targeted in the anticodon itself, we observe phage counter-selection of targeted isoacceptor tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Importance The presence of tRNAs in phages was discovered more than 50 years ago and their function has been debated ever since. Here, we propose that phage tRNAs counteract the tRNAse activities of the host, which may represent a depletion strategy of essential cellular components to stop translation and thereby phage infection. General Microbiology phage tRNAs mycobacteria tRNAses anticodon nucleases Figures Figure 1 Background Transfer RNAs (tRNAs) were first discovered in the 1950s (Kresge et al., 2005 ) and have since been found to play a vital role in the central dogma of molecular biology in all living systems (Crick, 1980). During the 1960s, tRNAs were also reported in viruses of bacteria (phages) (Weiss et al., 1968 ). We now know that phage-encoded tRNAs are widespread (Bailley-Bechet et al. , 2007). Multiple hypotheses have been proposed for the role of these phage-encoded tRNAs. The most established being codon compensation, where codons rarely used by the host but necessary to the phage are supplemented by the tRNAs carried by the phage (Bailly-Bechet et al., 2007 ). Why phages are pushed towards these alternative codons is generally believed to be a side effect of differences in the GC content of phage and host (Bailly-Bechet et al., 2007 ; Lucks et al., 2008 ; Limor-Waisberg et al., 2011 ). A recent study by Yang et al. ( 2021 ) may have hinted at an additional factor: phage tRNAs represent a means to counteract the depletion of host tRNAs that occurs as a general response to phage infection (Thompson & Parker, 2009 ; Yang et al., 2021 ; Jain et al., 2021 ; Amitsur et al., 1989 ). However, it remains unclear how phage tRNAs avoid being degraded by the same mechanism that results in host tRNA depletion during phage infection. Hypothesis We hypothesize that the tRNAs encoded by phages are insensitive to tRNA anticodon nuclease activity, preventing depletion of the tRNA pool during phage infection. To investigate this hypothesis, we analyzed the tRNAs encoded by a large and well-characterized dataset of tRNA-rich bacteriophages (33 tRNAs per phage on average) that infect mycobacteria: mycobacteriophage cluster C1 (Russell & Hatfull, 2017 ) (Fig. 1 A,B). The existence of these tRNA-rich phages coincides with a high abundance of tRNA nucleases (tRNAses), including the well-characterized VapCs, MazFs, and RelEs in Mycobacterium (Winther et al., 2016 ; Chauhan et al., 2022 ; Cruz et al., 2015 ; Cintrón et al., 2019 ; Barth et al., 2021 ; Pedersen et al., 2003 ). A subset of these tRNAses target the tRNA anticodon loop and are activated upon a variety of stress responses, including phage infection (Calcuttawala et al., 2022 ). When activated, these anticodon nucleases cleave specific tRNAs in conserved regions within the anticodon loop to inactivate these tRNAs and thereby regulate protein translation of the host (Winther et al., 2016 ). The cleavage region within the tRNA anticodon loop is sequence-dependent and highly specific for the type of tRNA. Mutations within the recognition and cleavage site in the anticodon loop have been found to cause insensitivity to these anticodon nucleases (Winther et al., 2016 ; Cruz et al., 2015 ). We compared the tRNAs encoded by phages with those of their host and observed all 10 phage-encoded tRNAs that are known to be targeted by anticodon nucleases to contain anticodon loop mutations (Winther et al., 2016 ; Cruz et al., 2015 ; Chauhan et al., 2022 ), reinforcing the idea that phage-encoded tRNAs are likely insensitive to cleavage (Fig. 1 C). We hypothesize that these phage tRNAs represent a means to counteract the depletion of tRNAs by anticodon nucleases during phage infection, allowing the phage to translate its proteins and complete its infection cycle (Fig. 1 D). Supporting the selective pressure of the host-encoded tRNA nucleases, we also observed a strong counter-selection for tRNAs that are cleaved in the anticodon itself (Table S1). This is the case for the majority of the serine-coding tRNAs that are cleaved at the GA site within the anticodon: tRNA-Ser(g ga ), tRNA-Ser(t ga ), tRNA-Ser(c ga ), and tRNA-Ser(a ga ) (Winther et al., 2016 ). In this instance, the phage encodes an isoacceptor tRNA that is not targeted (tRNA-Ser(gct)) to carry out translation independent from cleaved serine isoacceptor tRNAs. We observed the same counter-selection for the UAN anticodons, which are known targets of RelE in E. coli (Pedersen et al., 2003 ). Interestingly, we observed that phage genes do not avoid codons of cleaved tRNAs, nor do they have a preference for codons with nuclease insensitivity (Welch Two Sample t-test, t = 0.53848, df = 41.583, p-value > 0.05), suggesting that the selection of phage tRNAs is only determined by their insensitivity to tRNAses and not by codon usage. Altogether, our observations support the hypothesis that phage tRNAs are selected to be insensitive to anticodon nucleases to counteract tRNA-depletion strategies of the host that limit phage propagation. We expect that our hypothesis may be extended outside of Mycobacteria as phage tRNAs and host tRNAses are widespread (Ogawa et al., 2006 ; Covard & Lazdunski, 1979; Jones et al., 2017 ). Implications We argue that phage-encoded tRNAs escape targeting by host tRNAses, which can be helpful in selecting or engineering bacteriophages capable of infecting hosts containing anticodon nucleases. Method All C1 cluster mycobacteriophage genomes were downloaded from https://phagesdb.org/ on the 1st of September 2022. tRNAs were annotated using Aragorn (Laslett & Canback, 2004 ) and tRNAscan-SE (Chan et al. , 2019), and were further analyzed using MXfold2 (Sato et al., 2021 ). Cusp was used to calculate the codon frequency (Rice et al., 2000 ). Declarations Competing interests: The authors declare no competing interests. Acknowledgments: This work was supported by grants from the European Research Council (ERC) CoG under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 101003229) and the Netherlands Organisation for Scientific Research (NWO VICI; VI.C.192.027). References Amitsur, M., Morad, I., & Kaufmann, G. (1989). In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X. The EMBO Journal, 8 (8), 2411–2415. Bailly-Bechet, M., Vergassola, M., & Rocha, E. (2007). Causes for the intriguing presence of tRNAs in phages. Genome Research 17 (10), 1486–1495. Barth, V. C., Chauhan, U., Zeng, J., Su, X., Zheng, H., Husson, R. N., & Woychik, N. A. (2021). Mycobacterium tuberculosis VapC4 toxin engages small ORFs to initiate an integrated oxidative and copper stress response. Proceedings of the National Academy of Sciences , 118 (32), e2022136118. Calcuttawala, F., Shaw, R., Sarbajna, A., Dutta, M., Sinha, S., & K. Das Gupta, S. (2022). Apoptosis like symptoms associated with abortive infection of Mycobacterium smegmatis by mycobacteriophage D29. Plos one, 17 (5), e0259480. Cavard, D., & Lazdunski, C. (1979). Interaction of colicin E4 with specific receptor sites mediates its cleavage into two fragments inactive towards whole cells. European Journal of Biochemistry, 96 (3), 525–533. Chan, P. P., & Lowe, T. M. (2019). tRNAscan-SE: searching for tRNA genes in genomic sequences. In Gene prediction (pp. 1–14). Humana, New York, NY. Chauhan, U., Barth, V. C., & Woychik, N. A. (2022). tRNAfMet Inactivating Mycobacterium tuberculosis VapBC Toxin-Antitoxin Systems as Therapeutic Targets. Antimicrobial Agents and Chemotherapy, e01896-21. Cintrón, M., Zeng, J. M., Barth, V. C., Cruz, J. W., Husson, R. N., & Woychik, N. A. (2019). Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host. Scientific reports, 9 (1), 1–14. Crick, F. (1970). Central dogma of molecular biology. Nature, 227 (5258), 561–563. Cruz, J. W., Sharp, J. D., Hoffer, E. D., Maehigashi, T., Vvedenskaya, I. O., Konkimalla, A., … Woychik, N. A. (2015). Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nature communications, 6 (1), 1–12. Jain, I., Kolesnik, M., Minakhin, L., Morozova, N., Shiriaeva, A., Kirillov, A., … Semenova, E. (2021). tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector. bioRxiv . Jones, A. M., Garza-Sánchez, F., So, J., Hayes, C. S., & Low, D. A. (2017). Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors. Proceedings of the National Academy of Sciences , 114 (10), E1951-E1957. Kresge, N., Simoni, R. D., & Hill, R. L. (2005). The discovery of tRNA by Paul C. Zamecnik. Journal of Biological Chemistry, 280 (40), e37-e37. Laslett, D., & Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic acids research, 32 (1), 11–16. Limor-Waisberg, K., Carmi, A., Scherz, A., Pilpel, Y., & Furman, I. (2011). Specialization versus adaptation: two strategies employed by cyanophages to enhance their translation efficiencies. Nucleic acids research, 39 (14), 6016–6028. Lucks, J. B., Nelson, D. R., Kudla, G. R., & Plotkin, J. B. (2008). Genome landscapes and bacteriophage codon usage. PLoS computational biology, 4 (2), e1000001. Pedersen, K., Zavialov, A. V., Pavlov, M. Y., Elf, J., Gerdes, K., & Ehrenberg, M. (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131–140. Rice, P., Longden, I., & Bleasby, A. (2000). EMBOSS: the European molecular biology open software suite. Trends in genetics, 16 (6), 276–277. Russell, D. A., & Hatfull, G. F. (2017). PhagesDB: the actinobacteriophage database. Bioinformatics, 33 (5), 784–786. Sato, K., Akiyama, M., & Sakakibara, Y. (2021). RNA secondary structure prediction using deep learning with thermodynamic integration. Nature communications, 12 (1), 1–9. Thompson, D. M., & Parker, R. (2009). Stressing out over tRNA cleavage. Cell, 138 (2), 215–219. Ogawa, T., Inoue, S., Yajima, S., Hidaka, M., & Masaki, H. (2006). Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease. Nucleic acids research, 34 (21), 6065–6073. Weiss, S. B., Hsu, W. T., Foft, J. W., & Scherberg, N. H. (1968). Transfer RNA coded by the T4 bacteriophage genome. Proceedings of the National Academy of Sciences of the United States of America, 61 (1), 114. Winther, K., Tree, J. J., Tollervey, D., & Gerdes, K. (2016). VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation. Nucleic acids research, 44 (20), 9860–9871. Yang, J. Y., Fang, W., Miranda-Sanchez, F., Brown, J. M., Kauffman, K. M., Acevero, C. M., … Kelly, L. (2021). Degradation of host translational machinery drives tRNA acquisition in viruses. Cell Systems, 12(8), 771–779. Supplementary Files TableS1.xlsx Table S1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2166710","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":144319144,"identity":"edb54728-4b23-40a2-b2cb-41e4d1c439e4","order_by":0,"name":"Daan F. van den Berg","email":"","orcid":"https://orcid.org/0000-0002-2217-4074","institution":"Department of Bionanoscience, Delft University of Technology, Delft, Netherlands","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daan","middleName":"F. van den","lastName":"Berg","suffix":""},{"id":144320206,"identity":"ab6cefcd-853f-457c-be7a-204dc2ffb6a3","order_by":1,"name":"Baltus A. van der Steen","email":"","orcid":"https://orcid.org/0000-0001-8193-4814","institution":"Department of Bionanoscience, Delft University of Technology, Delft, Netherlands","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Baltus","middleName":"A. van der","lastName":"Steen","suffix":""},{"id":144320207,"identity":"c8bf0350-e0c7-4407-b753-99ccfe2e022d","order_by":2,"name":"Ana Rita Costa","email":"","orcid":"https://orcid.org/0000-0001-6749-6408","institution":"Department of Bionanoscience, Delft University of Technology, Delft, Netherlands","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Rita","lastName":"Costa","suffix":""},{"id":144320208,"identity":"d113090e-1347-42ea-8baa-4d310e368182","order_by":3,"name":"Stan J. J. Brouns","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYJACZih1AEhIyBBWzwbXwpYA0sJDihYeAzBJUIf8/OZnjwv32CRu5z/z+dWNGgseBvbDRzfg02JwjM3ceMaztMSdM3K3WeccAzqMJy3tBl4tbAxm0jwHDiduuMG7zTiHDahFgscMrxb5NvZvEC3nzzwzzvlHhBaGYzxQWw7kMD/ObSNCi8GxnDLpGQfSjDfcSDNjzu2T4GEj5Bf55uPbpAsO2MhuOH/48eecb3Vy/OyHj+F3GBJgkwCTxCoHAeYPpKgeBaNgFIyCkQMAdZdGTEOwmnkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9573-1724","institution":"Department of Bionanoscience, Delft University of Technology, Delft, Netherlands","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Stan","middleName":"J. J.","lastName":"Brouns","suffix":""}],"badges":[],"createdAt":"2022-10-14 14:33:02","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-2166710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2166710/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28868688,"identity":"ca0045a3-b6a0-4462-b4d3-f176bf46e75f","added_by":"auto","created_at":"2022-11-09 18:03:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1094358,"visible":true,"origin":"","legend":"\u003cp\u003ePhage tRNAs are predicted to be anticodon nuclease resistant.\u003cstrong\u003e \u003c/strong\u003e(A) The genomic context of the tRNA clusters containing 36 tRNAs present in C1 mycobacteriophage Rizal (Russell \u0026amp; Hatfull, 2017). (B) Prevalence of individual phage-encoded tRNAs in the C1 mycobacteriophage cluster, composed of 161 phages. (C) Mutations in the anticodon-loop of phage tRNAs in comparison to host tRNAs, located in the cleavage site of anticodon nucleases. (D) Proposed mechanism of action of phage tRNAs. During phage infection, tRNAses are activated and deplete the host tRNA pool via tRNA cleavage to prevent phage propagation. Phage tRNAs are insensitive to cleavage and refill the tRNA pool allowing the phage to propagate.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2166710/v1/4131fac86d6c0037f31f2b6b.png"},{"id":28868700,"identity":"1e714942-56c3-40e2-853c-ccc64d3746c5","added_by":"auto","created_at":"2022-11-09 18:04:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":738648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2166710/v1/01c053dd-e423-4c43-8afe-dcffdd085a3c.pdf"},{"id":28868687,"identity":"ab69ed85-6371-461b-9e7c-8cb8cedfc238","added_by":"auto","created_at":"2022-11-09 18:03:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28342,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2166710/v1/7d153b4ba1ac54243fb837c4.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePhage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eTransfer RNAs (tRNAs) were first discovered in the 1950s (Kresge et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and have since been found to play a vital role in the central dogma of molecular biology in all living systems (Crick, 1980). During the 1960s, tRNAs were also reported in viruses of bacteria (phages) (Weiss et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). We now know that phage-encoded tRNAs are widespread (Bailley-Bechet \u003cem\u003eet al.\u003c/em\u003e, 2007). Multiple hypotheses have been proposed for the role of these phage-encoded tRNAs. The most established being codon compensation, where codons rarely used by the host but necessary to the phage are supplemented by the tRNAs carried by the phage (Bailly-Bechet et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Why phages are pushed towards these alternative codons is generally believed to be a side effect of differences in the GC content of phage and host (Bailly-Bechet et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lucks et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Limor-Waisberg et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A recent study by Yang et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) may have hinted at an additional factor: phage tRNAs represent a means to counteract the depletion of host tRNAs that occurs as a general response to phage infection (Thompson \u0026amp; Parker, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jain et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Amitsur et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). However, it remains unclear how phage tRNAs avoid being degraded by the same mechanism that results in host tRNA depletion during phage infection.\u003c/p\u003e"},{"header":"Hypothesis","content":"\u003cp\u003eWe hypothesize that the tRNAs encoded by phages are insensitive to tRNA anticodon nuclease activity, preventing depletion of the tRNA pool during phage infection. To investigate this hypothesis, we analyzed the tRNAs encoded by a large and well-characterized dataset of tRNA-rich bacteriophages (33 tRNAs per phage on average) that infect mycobacteria: mycobacteriophage cluster C1 (Russell \u0026amp; Hatfull, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). The existence of these tRNA-rich phages coincides with a high abundance of tRNA nucleases (tRNAses), including the well-characterized VapCs, MazFs, and RelEs in Mycobacterium (Winther et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chauhan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cruz et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cintr\u0026oacute;n et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barth et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pedersen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A subset of these tRNAses target the tRNA anticodon loop and are activated upon a variety of stress responses, including phage infection (Calcuttawala et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When activated, these anticodon nucleases cleave specific tRNAs in conserved regions within the anticodon loop to inactivate these tRNAs and thereby regulate protein translation of the host (Winther et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The cleavage region within the tRNA anticodon loop is sequence-dependent and highly specific for the type of tRNA. Mutations within the recognition and cleavage site in the anticodon loop have been found to cause insensitivity to these anticodon nucleases (Winther et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cruz et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We compared the tRNAs encoded by phages with those of their host and observed all 10 phage-encoded tRNAs that are known to be targeted by anticodon nucleases to contain anticodon loop mutations (Winther et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cruz et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chauhan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), reinforcing the idea that phage-encoded tRNAs are likely insensitive to cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We hypothesize that these phage tRNAs represent a means to counteract the depletion of tRNAs by anticodon nucleases during phage infection, allowing the phage to translate its proteins and complete its infection cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSupporting the selective pressure of the host-encoded tRNA nucleases, we also observed a strong counter-selection for tRNAs that are cleaved in the anticodon itself (Table S1). This is the case for the majority of the serine-coding tRNAs that are cleaved at the GA site within the anticodon: tRNA-Ser(g\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ega\u003c/span\u003e), tRNA-Ser(t\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ega\u003c/span\u003e), tRNA-Ser(c\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ega\u003c/span\u003e), and tRNA-Ser(a\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ega\u003c/span\u003e) (Winther et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this instance, the phage encodes an isoacceptor tRNA that is not targeted (tRNA-Ser(gct)) to carry out translation independent from cleaved serine isoacceptor tRNAs. We observed the same counter-selection for the UAN anticodons, which are known targets of RelE in \u003cem\u003eE. coli\u003c/em\u003e (Pedersen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Interestingly, we observed that phage genes do not avoid codons of cleaved tRNAs, nor do they have a preference for codons with nuclease insensitivity (Welch Two Sample t-test, t\u0026thinsp;=\u0026thinsp;0.53848, df\u0026thinsp;=\u0026thinsp;41.583, p-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting that the selection of phage tRNAs is only determined by their insensitivity to tRNAses and not by codon usage. Altogether, our observations support the hypothesis that phage tRNAs are selected to be insensitive to anticodon nucleases to counteract tRNA-depletion strategies of the host that limit phage propagation. We expect that our hypothesis may be extended outside of Mycobacteria as phage tRNAs and host tRNAses are widespread (Ogawa et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Covard \u0026amp; Lazdunski, 1979; Jones et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e"},{"header":"Implications","content":"\u003cp\u003eWe argue that phage-encoded tRNAs escape targeting by host tRNAses, which can be helpful in selecting or engineering bacteriophages capable of infecting hosts containing anticodon nucleases.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003eAll C1 cluster mycobacteriophage genomes were downloaded from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phagesdb.org/\u003c/span\u003e\u003cspan address=\"https://phagesdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e on the 1st of September 2022. tRNAs were annotated using Aragorn (Laslett \u0026amp; Canback, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and tRNAscan-SE (Chan \u003cem\u003eet al.\u003c/em\u003e, 2019), and were further analyzed using MXfold2 (Sato et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cusp was used to calculate the codon frequency (Rice et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the European Research Council (ERC) CoG under the European Union\u0026rsquo;s Horizon 2020 research and innovation program (grant agreement No. 101003229) and the Netherlands Organisation for Scientific Research (NWO VICI; VI.C.192.027).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmitsur, M., Morad, I., \u0026amp; Kaufmann, G. (1989). In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X. 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Proceedings of the National Academy of Sciences of the United States of America, \u003cem\u003e61\u003c/em\u003e(1), 114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinther, K., Tree, J. J., Tollervey, D., \u0026amp; Gerdes, K. (2016). VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation. Nucleic acids research, \u003cem\u003e44\u003c/em\u003e(20), 9860\u0026ndash;9871.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J. Y., Fang, W., Miranda-Sanchez, F., Brown, J. M., Kauffman, K. M., Acevero, C. M., \u0026hellip; Kelly, L. (2021). Degradation of host translational machinery drives tRNA acquisition in viruses. Cell Systems, 12(8), 771\u0026ndash;779.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"3c528757-93fd-4640-8807-fbca9359a947","identifier":"10.13039/501100000781","name":"European Research Council","awardNumber":"101003229","order_by":0},{"identity":"8bb41610-f9a8-4e66-b789-8b3ec2b8f9e1","identifier":"10.13039/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","awardNumber":"VI.C.192.027","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Delft University of Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"phage, tRNAs, mycobacteria, tRNAses, anticodon nucleases","lastPublishedDoi":"10.21203/rs.3.rs-2166710/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2166710/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003etRNAs in bacteriophage genomes are widespread across bacterial genera, but their exact function has remained unclear for more than 50 years. Multiple hypotheses have been proposed, with the most established being codon compensation, where codons more rarely used by the host but necessary for the phage are supplemented by tRNAs encoded by the phage. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs are a means to counteract the tRNA-depleting strategies of the host to defend from viral infection. Based on mutational patterns of tRNA anticodon loops, we predict that phage tRNAs are insensitive to the host tRNAses. For tRNAs targeted in the anticodon itself, we observe phage counter-selection of targeted isoacceptor tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases.\u003c/p\u003e \u003cp\u003eImportance\u003c/p\u003e \u003cp\u003eThe presence of tRNAs in phages was discovered more than 50 years ago and their function has been debated ever since. Here, we propose that phage tRNAs counteract the tRNAse activities of the host, which may represent a depletion strategy of essential cellular components to stop translation and thereby phage infection.\u003c/p\u003e","manuscriptTitle":"Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-09 18:03:51","doi":"10.21203/rs.3.rs-2166710/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7387761f-6a6f-4652-a8d9-77d399dc2e86","owner":[],"postedDate":"November 9th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":16253699,"name":"General Microbiology"}],"tags":[],"updatedAt":"2022-11-09T18:03:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-09 18:03:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2166710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2166710","identity":"rs-2166710","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}