Transfer RNA-Derived Small RNAs: Another Layer of Gene Regulation and Novel Targets for Disease Therapeutics
Authors: Hak Kyun Kim, Ji-Hyun Yeom, Mark A. Kay
Journal: Molecular Therapy (2020)

Decades after identification as essential for protein synthesis, transfer RNAs (tRNAs) have been implicated in various cellular processes beyond translation. tRNA-derived small RNAs (tsRNAs), referred to as tRNA-derived fragments (tRFs) or tRNA-derived, stress-induced RNAs (tiRNAs), are produced by cleavage at different sites from mature or pre-tRNAs. They are classified into six major types representing potentially thousands of unique sequences and have been implicated to play a wide variety of regulatory roles in maintaining normal homeostasis, cancer cell viability, tumorigenesis, ribosome biogenesis, chromatin remodeling, translational regulation, intergenerational inheritance, retrotransposon regulation, and viral replication. However, the detailed mechanisms governing these processes remain unknown. Aberrant expression of tsRNAs is found in various human disease conditions, suggesting that a further understanding of the regulatory role of tsRNAs will assist in identifying novel biomarkers, potential therapeutic targets, and gene-regulatory tools. Here, we highlight the classification, biogenesis, and biological role of tsRNAs in regulatory mechanisms of normal and disease states. Decades after identification as essential for protein synthesis, transfer RNAs (tRNAs) have been implicated in various cellular processes beyond translation. tRNA-derived small RNAs (tsRNAs), referred to as tRNA-derived fragments (tRFs) or tRNA-derived, stress-induced RNAs (tiRNAs), are produced by cleavage at different sites from mature or pre-tRNAs. They are classified into six major types representing potentially thousands of unique sequences and have been implicated to play a wide variety of regulatory roles in maintaining normal homeostasis, cancer cell viability, tumorigenesis, ribosome biogenesis, chromatin remodeling, translational regulation, intergenerational inheritance, retrotransposon regulation, and viral replication. However, the detailed mechanisms governing these processes remain unknown. Aberrant expression of tsRNAs is found in various human disease conditions, suggesting that a further understanding of the regulatory role of tsRNAs will assist in identifying novel biomarkers, potential therapeutic targets, and gene-regulatory tools. Here, we highlight the classification, biogenesis, and biological role of tsRNAs in regulatory mechanisms of normal and disease states. Transfer RNAs (tRNAs) are non-coding RNAs of 76–93 nt in length essential for mRNA translation. They decode genetic information into proteins by transferring amino acids to the growing polypeptide chains that are synthesized on ribosomes.1Rodnina M.V. Wintermeyer W. The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation.Biochem. Soc. Trans. 2011; 39: 658-662Crossref PubMed Scopus (76) Google Scholar tRNAs are processed from pre-tRNAs transcribed by RNA polymerase III (RNA pol III).2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (395) Google Scholar During maturation, pre-tRNAs undergo several modifications, such as the removal of the 5′ leader and 3′ trailer sequences, splicing, post-transcriptional modification, and addition of a CCA sequence at the 3′ end of mature tRNA. tRNAs are, by far, the most abundant RNA species, and their sequences are highly conserved across species. They are grouped into 64 types according to their anti-codon sequence. More than 500 loci in human genomes have hitherto been identified, and over 400 tRNA genes have unique sequences.3Chan P.P. Lowe T.M. GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.Nucleic Acids Res. 2016; 44: D184-D189Crossref PubMed Scopus (222) Google Scholar In addition, there are over 600 tRNA pseudogenes, and tRNA lookalikes in the human nuclear genome and their functional significance remain unknown.3Chan P.P. Lowe T.M. GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.Nucleic Acids Res. 2016; 44: D184-D189Crossref PubMed Scopus (222) Google Scholar,4Telonis A.G. Loher P. Kirino Y. Rigoutsos I. Nuclear and mitochondrial tRNA-lookalikes in the human genome.Front. Genet. 2014; 5: 344Crossref PubMed Scopus (17) Google Scholar For decades, tRNAs have been mostly considered to play a role in protein synthesis. However, it is becoming increasingly clear that tRNAs have various additional roles involved in cell proliferation, tumor metastasis, and neuronal homeostasis.5Schimmel P. The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.Nat. Rev. Mol. Cell Biol. 2018; 19: 45-58Crossref PubMed Scopus (90) Google Scholar The first tRNA mutations related with human disease were identified in 1990.6Kobayashi Y. Momoi M.Y. Tominaga K. Momoi T. Nihei K. Yanagisawa M. Kagawa Y. Ohta S. A point mutation in the mitochondrial tRNA(Leu)(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes).Biochem. Biophys. Res. Commun. 1990; 173: 816-822Crossref PubMed Scopus (296) Google Scholar,7Shoffner J.M. Lott M.T. Lezza A.M. Seibel P. Ballinger S.W. Wallace D.C. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation.Cell. 1990; 61: 931-937Abstract Full Text PDF PubMed Scopus (1179) Google Scholar These mutations were found in mitochondrial-expressed tRNAs and result in neuromuscular phenotypes. In contrast, no human disease has been associated with a cytoplasmic tRNA mutation, per se, but mutations in genes related to post-transcriptional modification of tRNAs and cytoplasmic tRNA-related protein partners, including splicing factors and aminoacyl tRNA-synthetase (ARSs), are strongly associated with certain human diseases.8Abbott J.A. Francklyn C.S. Robey-Bond S.M. Transfer RNA and human disease.Front. Genet. 2014; 5: 158Crossref PubMed Scopus (106) Google Scholar For example, mutations in the tRNA-splicing endonuclease complex (TSEN2, TSEN15, TSEN34, and TSEN54) were identified in PCH (pontocerebellar hypoplasia) type 2 and 4 patients, of which brain tissue showed immature development of the cerebellum and other structural defects.9Budde B.S. Namavar Y. Barth P.G. Poll-The B.T. Nürnberg G. Becker C. van Ruissen F. Weterman M.A. Fluiter K. te Beek E.T. et al.tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia.Nat. Genet. 2008; 40: 1113-1118Crossref PubMed Scopus (147) Google Scholar tRNA-derived small RNAs (tsRNAs) were first discovered in the urine of cancer patients in the 1970s.10Borek E. Baliga B.S. Gehrke C.W. Kuo C.W. Belman S. Troll W. Waalkes T.P. High turnover rate of transfer RNA in tumor tissue.Cancer Res. 1977; 37: 3362-3366PubMed Google Scholar,11Speer J. Gehrke C.W. Kuo K.C. Waalkes T.P. Borek E. tRNA breakdown products as markers for cancer.Cancer. 1979; 44: 2120-2123Crossref PubMed Scopus (128) Google Scholar At the time, these RNAs were considered to be degradation products. With the advent of next-generation, high-throughput sequencing (NGS), the sequencing reads corresponding to tsRNAs were often discarded as degradation products or sequencing artifacts. However, just over a decade ago, three groups revealed that tsRNAs have biological roles.12Lee Y.S. Shibata Y. Malhotra A. Dutta A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs).Genes Dev. 2009; 23: 2639-2649Crossref PubMed Scopus (538) Google Scholar, 13Yamasaki S. Ivanov P. Hu G.-F. Anderson P. Angiogenin cleaves tRNA and promotes stress-induced translational repression.J. Cell Biol. 2009; 185: 35-42Crossref PubMed Scopus (419) Google Scholar, 14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar A growing body of evidence has demonstrated that tsRNA expression is not correlated with cognate tRNA abundance and they are processed at specific cleavage sites of mature and pre-tRNAs.15Kumar P. Anaya J. Mudunuri S.B. Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.BMC Biol. 2014; 12: 78Crossref PubMed Scopus (172) Google Scholar Interestingly, tRNAs are differentially expressed in a variety of tissues, cancers, and during normal growth and development, including cellular differentiation.16Gingold H. Tehler D. Christoffersen N.R. Nielsen M.M. Asmar F. Kooistra S.M. Christophersen N.S. Christensen L.L. Borre M. Sørensen K.D. et al.A dual program for translation regulation in cellular proliferation and differentiation.Cell. 2014; 158: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar The same tRNA isodecoder genes (containing the same anti-codon sequence but with differences in each body sequence) are differentially expressed in several cases,17Dittmar K.A. Goodenbour J.M. Pan T. Tissue-specific differences in human transfer RNA expression.PLoS Genet. 2006; 2: e221Crossref PubMed Scopus (322) Google Scholar which suggests that the differential expression of each tRNA gene may be related to noncanonical tRNA functions (e.g., tsRNA). Thus far, six major types of tsRNAs have been identified in various organisms, from humans to Escherichia. coli (E. coli), and they have been implicated in various diseases with distinct mechanisms. The fact that there are hundreds of tRNA genes that can be cleaved at specific sites to produce many different types and lengths of small RNAs suggests that thousands of unique and de novo non-coding RNAs can potentially be generated in a cell. Together, all of these findings demonstrate that tsRNAs are not merely degradation products but are generated in a precise and regulated manner. Throughout this review, we will provide an overview of tsRNA classification and biogenesis while delineating known tsRNA-mediated regulatory mechanisms. In addition, we describe potential tsRNA associations with disease-related processes, when known, and how these interactions could empower new disease biomarkers or be manipulated for novel treatments. tsRNAs are also called tRF (tRNA-derived RNA fragment), tDRs (tRNA-derived small RNAs), and tiRNAs (tRNA-derived stress-induced RNAs).12Lee Y.S. Shibata Y. Malhotra A. Dutta A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs).Genes Dev. 2009; 23: 2639-2649Crossref PubMed Scopus (538) Google Scholar, 13Yamasaki S. Ivanov P. Hu G.-F. Anderson P. Angiogenin cleaves tRNA and promotes stress-induced translational repression.J. Cell Biol. 2009; 185: 35-42Crossref PubMed Scopus (419) Google Scholar, 14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar tsRNAs are differently named according to length and cleavage position on tRNA or pre-tRNA. tsRNAs of 18–30 nt in length are categorized as either type I or type II, according to the cleaved position on mature tRNA and pre-tRNA, respectively (Figure 1). Type I tsRNAs are further classified into two subgroups: 5′tsRNA (tRF-5 or 5′tRF) and 3′tsRNA (tRF-3 or 3′tRF), which are derived from the 5′ and 3′ ends of mature tRNA, respectively.14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar NGS data show that 5′tsRNAs from HEK293 cells mainly occur at lengths of 14–16 nt (tRF-5a), 22–24 nt (tRF-5b), and 28–30 nt (tRF-5c), which begin at the 5′ termini of mature tRNAs and end at a D-loop (tRF-5a) or at a stem region between the D-loop and the anti-codon loop (tRF-5b and tRF-5c) of a mature tRNA.15Kumar P. Anaya J. Mudunuri S.B. Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.BMC Biol. 2014; 12: 78Crossref PubMed Scopus (172) Google Scholar Other lengths of 5′tsRNAs were also identified in prostate cancer cells and Haloferax volcanii,18Gebetsberger J. Zywicki M. Künzi A. Polacek N. tRNA-derived fragments target the ribosome and function as regulatory non-coding RNA in Haloferax volcanii.Archaea. 2012; 2012: 260909Crossref PubMed Scopus (126) Google Scholar indicating that the length of 5′tsRNA might be more heterogeneous than expected, depending on the cell type and species. On the other hand, 3′tsRNAs (3′tRF) are uniformly present in the 18- to 22-nt forms throughout the three domains of life.15Kumar P. Anaya J. Mudunuri S.B. Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.BMC Biol. 2014; 12: 78Crossref PubMed Scopus (172) Google Scholar Their 5′ ends reside in the T-loop, and the 3′ end corresponds with the 3′ terminus of mature tRNA, which all ends in CCA. Interestingly, most 22-nt 3′tsRNAs contain an N1-methyl-adenosine (m1A58) at the 58th nt in the T-loop (corresponding to the 19th nt in the tsRNA) and a pseudouridine at or near their 5′ termini,19Rozenski J. Crain P.F. McCloskey J.A. The RNA Modification Database: 1999 update.Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (343) Google Scholar,20Anderson J.T. Droogmans L. Biosynthesis and function of 1-methyladenosine in transfer RNA.Fine-Tuning of RNA Functions by Modification and Editing, H. Grosjean. Springer, 2005: 121-139Crossref Google Scholar perhaps because one or both features may play a role in the generation of this type of tsRNA. Type II tsRNAs (tRF-1 or 3′U tRF) 16–48 nt in length are processed from a 3′ trailer sequence of pre-tRNA that begins 1 to 2 nt downstream of the 3′ end of the tRNA genomic sequence and ends with a polyuridine sequence (UUUUU, UUCUU, GUCUU, or AUCUU), a RNA pol III termination signal.12Lee Y.S. Shibata Y. Malhotra A. Dutta A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs).Genes Dev. 2009; 23: 2639-2649Crossref PubMed Scopus (538) Google Scholar,14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar Because the positions of the termination signal vary for each tRNA transcript, the length of the type II tsRNAs are more heterogeneous than type I tsRNAs.14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar,15Kumar P. Anaya J. Mudunuri S.B. Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.BMC Biol. 2014; 12: 78Crossref PubMed Scopus (172) Google Scholar tsRNAs of length 30–40 nt are called "tRNA halves" because their lengths are almost half that of mature tRNA.21Lee S.R. Collins K. Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila.J. Biol. Chem. 2005; 280: 42744-42749Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar They are often referred to as "tiRNA" due to their stress-induced characteristic,13Yamasaki S. Ivanov P. Hu G.-F. Anderson P. Angiogenin cleaves tRNA and promotes stress-induced translational repression.J. Cell Biol. 2009; 185: 35-42Crossref PubMed Scopus (419) Google Scholar and some of them are called "SHOT-RNAs" (sex-hormone-dependent tRNA-derived RNA) because they are highly expressed in hormone-dependent cancers.22Honda S. Loher P. Shigematsu M. Palazzo J.P. Suzuki R. Imoto I. Rigoutsos I. Kirino Y. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers.Proc. Natl. Acad. Sci. USA. 2015; 112: E3816-E3825Crossref PubMed Scopus (119) Google Scholar They are grouped into 5′tRNA halves (5′tiRNA) and 3′tRNA halves (3′tiRNA) (Figure 1). The 5′ end of the former corresponds with the 5′ end of the mature corresponding tRNA, although the 3′ end is in the anti-codon loop. The latter begins in the anti-codon loop and ends with the 3′ terminus of the mature tRNA (Figure 1).13Yamasaki S. Ivanov P. Hu G.-F. Anderson P. Angiogenin cleaves tRNA and promotes stress-induced translational repression.J. Cell Biol. 2009; 185: 35-42Crossref PubMed Scopus (419) Google Scholar,22Honda S. Loher P. Shigematsu M. Palazzo J.P. Suzuki R. Imoto I. Rigoutsos I. Kirino Y. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers.Proc. Natl. Acad. Sci. USA. 2015; 112: E3816-E3825Crossref PubMed Scopus (119) Google Scholar There are two additional types of tsRNAs that are not included in the classes described above. i-tRF,23Telonis A.G. Loher P. Honda S. Jing Y. Palazzo J. Kirino Y. Rigoutsos I. Dissecting tRNA-derived fragment complexities using personalized transcriptomes reveals novel fragment classes and unexpected dependencies.Oncotarget. 2015; 6: 24797-24822Crossref PubMed Scopus (33) Google Scholar referred to as tRF-2,24Kumar P. Kuscu C. Dutta A. Biogenesis and function of transfer RNA-related fragments (tRFs).Trends Biochem. Sci. 2016; 41: 679-689Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar with a variable length is derived from the internal region of mature tRNA straddling the anti-codon region (Figure 1).23Telonis A.G. Loher P. Honda S. Jing Y. Palazzo J. Kirino Y. Rigoutsos I. Dissecting tRNA-derived fragment complexities using personalized transcriptomes reveals novel fragment classes and unexpected dependencies.Oncotarget. 2015; 6: 24797-24822Crossref PubMed Scopus (33) Google Scholar,25Goodarzi H. Liu X. Nguyen H.C.B. Zhang S. Fish L. Tavazoie S.F. Endogenous tRNA-derived fragments suppress breast cancer progression via YBX1 displacement.Cell. 2015; 161: 790-802Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar The other type begins at the 5′ end of the leader sequences in pre-tRNA and ends in the 3′ terminus of the 5′ exon in the anti-codon loop after removal of introns, which is implicated in neuronal degeneration (Figure 1).26Hanada T. Weitzer S. Mair B. Bernreuther C. Wainger B.J. Ichida J. Hanada R. Orthofer M. Cronin S.J. Komnenovic V. et al.CLP1 links tRNA metabolism to progressive motor-neuron loss.Nature. 2013; 495: 474-480Crossref PubMed Scopus (116) Google Scholar The information of varied tsRNAs has been retrieved from numerous deep-sequencing analyses and have been made available on public websites: tRFdb (http://genome.bioch.virginia.edu/trfdb) and tRF2Cancer (http://rna.sysu.edu.cn/tRFfinder).27Kumar P. Mudunuri S.B. Anaya J. Dutta A. tRFdb: a database for transfer RNA fragments.Nucleic Acids Res. 2015; 43: D141-D145Crossref PubMed Scopus (74) Google Scholar,28Zheng L.-L. Xu W.-L. Liu S. Sun W.-J. Li J.-H. Wu J. Yang J.H. Qu L.H. tRF2Cancer: a web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.Nucleic Acids Res. 2016; 44: W185-W193Crossref PubMed Scopus (34) Google Scholar So far, the nomenclature for tsRNA has not been established. In order to intuitively understand which mature or pre-tRNAs generate the indicated tsRNAs in our text, we have added each tRNA name to the term "tsRNA" or "tRNA halves" according to their size and also include the names used in each study. Other tsRNAs not included in the six major subtypes described above will be referred to by the name used in each study. Because type I and II tsRNAs are primarily 22 nt in length and are also known to be 5′ phosphorylated and 3′ hydroxylated,14Haussecker D. Huang Y. Lau A. Parameswaran P. Fire A.Z. Kay M.A. Human tRNA-derived small RNAs in the global regulation of RNA silencing.RNA. 2010; 16: 673-695Crossref PubMed Scopus (382) Google Scholar,29Couvillion M.T. Sachidanandam R. Collins K. A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo.Genes Dev. 2010; 24: 2742-2747Crossref PubMed Scopus (86) Google Scholar which are characteristics of microRNAs (miRNAs), early investigation into the biogenesis of tsRNAs was focused on the involvement of Dicer or the microprocessor complex (Drosha/DGCR8), both of which are required for the biogenesis of miRNA.30Kim V.N. MicroRNA biogenesis: coordinated cropping and dicing.Nat. Rev. Mol. Cell Biol. 2005; 6: 376-385Crossref PubMed Scopus (1791) Google Scholar tsRNAs (3′ end of Ile tRNA precursor) in mouse embryonic stem cells (mESCs) were initially found to be processed by Dicer, but not DGCR8.31Babiarz J.E. Ruby J.G. Wang Y. Bartel D.P. Blelloch R. Mouse ES cells express endogenous shRNAs, siRNAs, and other microprocessor-independent, Dicer-dependent small RNAs.Genes Dev. 2008; 22: 2773-2785Crossref PubMed Scopus (552) Google Scholar The Ile tRNA precursor is predicted to form a long hairpin structure as an alternative form, which is considered to be a substrate of Dicer. In human cell lines, 19nt-Gln5′tsRNA and 22nt-GlyGCC3′tsRNA (CU1276) were also reported to be processed by Dicer.32Cole C. Sobala A. Lu C. Thatcher S.R. Bowman A. Brown J.W.S. Green P.J. Barton G.J. Hutvagner G. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs.RNA. 2009; 15: 2147-2160Crossref PubMed Scopus (360) Google Scholar,33Maute R.L. Schneider C. Sumazin P. Holmes A. Califano A. Basso K. Dalla-Favera R. tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma.Proc. Natl. Acad. Sci. USA. 2013; 110: 1404-1409Crossref PubMed Scopus (239) Google Scholar An 18-nt Lys3′tsRNA (PBSncRNA), which is highly expressed in human immunodeficiency virus (HIV)-1-infected MT4 cells, is processed by Dicer.34Yeung M.L. Bennasser Y. Watashi K. Le S.-Y. Houzet L. Jeang K.-T. Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid.Nucleic Acids Res. 2009; 37: 6575-6586Crossref PubMed Scopus (145) Google Scholar In contrast, NGS data analyses from Dicer or Dgcr8 knockout mESCs indicate that type I tsRNAs are not processed using a canonical miRNA pathway.35Li Z. Ender C. Meister G. Moore P.S. Chang Y. John B. Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs.Nucleic Acids Res. 2012; 40: 6787-6799Crossref PubMed Scopus (158) Google Scholar Dicer mutants in mouse, D. melanogaster, and S. pombe do not significantly change type I or II tsRNA expression.15Kumar P. Anaya J. Mudunuri S.B. Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.BMC Biol. 2014; 12: 78Crossref PubMed Scopus (172) Google Scholar Dicer knockout 293T cells also show no differences in tsRNA expression.36Kuscu C. Kumar P. Kiran M. Su Z. Malik A. Dutta A. tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner.RNA. 2018; 24: 1093-1105Crossref PubMed Scopus (66) Google Scholar At most, these data suggest that Dicer and the microprocessor complex may only process a small number of specific tRNAs and/or in a cell-type-specific manner during particular cellular events. There are many lines of evidence demonstrating that tsRNAs are processed by endonucleases other than the miRNA-processing enzymes. For example, LysTTT3′tsRNA is processed by other ribonucleases, including angiogenin, in an in vitro cleavage assay.35Li Z. Ender C. Meister G. Moore P.S. Chang Y. John B. Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs.Nucleic Acids Res. 2012; 40: 6787-6799Crossref PubMed Scopus (158) Google Scholar RNase T2 is responsible for the production of tsRNAs in length of 17–40 nt in plants. Human RNase T2 and yeast Rny1p, a member of the RNase T2 family, are also able to generate 17- to 40-nt tsRNAs.37Megel C. Hummel G. Lalande S. Ubrig E. Cognat V. Morelle G. Salinas-Giegé T. Duchêne A.M. Maréchal-Drouard L. Plant RNases T2, but not Dicer-like proteins, are major players of tRNA-derived fragments biogenesis.Nucleic Acids Res. 2019; 47: 941-952Crossref PubMed Scopus (20) Google Scholar Type II tsRNAs are also processed by different endonucleases. RNA pol III transcribes the pre-tRNA gene, which is composed of a 5′ leader, mature tRNA, and 3′ trailer sequence.2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (395) Google Scholar,38Phizicky E.M. 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A candidate prostate cancer susceptibility gene encodes tRNA 3′ processing endoribonuclease.Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (134) Google Scholar and a prostate cancer susceptibility gene, processes SerTGA type II tsRNA (tRF-1001) from pre-tRNA in a prostate cancer cell line.12Lee Y.S. Shibata Y. Malhotra A. Dutta A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs).Genes Dev. 2009; 23: 2639-2649Crossref PubMed Scopus (538) Google Scholar Overall, more work is required to further determine the full detailed mechanism by which type II tsRNAs are processed, the location(s) of processing (i.e., nuclear versus cytoplasmic), and how such processing may be linked to regulating their production. The biogenesis of tRNA halves 30–40 nt in length is more established than the 17- to 30-nt fragments. tRNA halves were initially found in E. coli and were generated by PrrC nuclease in response to bacteriophage infection.40Levitz R. Chapman D. Amitsur M. 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