The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

Abstract Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5′-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5′-tRNA halves as well, suggesting a previously uncharacterized link between 5′-tRNA halves and td-piRNAs. An increase in levels of the 5′-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5′-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to −1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs.

[1]  S. Sugano,et al.  The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes. , 2009, RNA.

[2]  Thurston H. Y. Dang,et al.  The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells , 2014, Nucleic acids research.

[3]  S. Kawaoka,et al.  3' end formation of PIWI-interacting RNAs in vitro. , 2011, Molecular cell.

[4]  N. Polacek,et al.  Slicing tRNAs to boost functional ncRNA diversity , 2013, RNA biology.

[5]  Izabela Makałowska,et al.  Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA(CAA)Leu , 2006, Nucleic acids research.

[6]  Rogerio Margis,et al.  Description of plant tRNA-derived RNA fragments (tRFs) associated with argonaute and identification of their putative targets , 2013, Biology Direct.

[7]  G. Barton,et al.  Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. , 2009, RNA.

[8]  S. Yamasaki,et al.  Angiogenin cleaves tRNA and promotes stress-induced translational repression , 2009, The Journal of cell biology.

[9]  Yoshihide Hayashizaki,et al.  Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin , 2011, RNA biology.

[10]  Lucian Ilie,et al.  SHRiMP2: Sensitive yet Practical Short Read Mapping , 2011, Bioinform..

[11]  Zissimos Mourelatos,et al.  Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini , 2007, Nature Structural &Molecular Biology.

[12]  Andrea Califano,et al.  tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma , 2013, Proceedings of the National Academy of Sciences.

[13]  Praveen Sethupathy,et al.  Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C , 2015, Scientific Reports.

[14]  R. Sachidanandam,et al.  piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire , 2015, Genes & development.

[15]  Natsuko Izumi,et al.  Identification and Functional Analysis of the Pre-piRNA 3′ Trimmer in Silkworms , 2016, Cell.

[16]  Sergey Steinberg,et al.  Compilation of tRNA sequences and sequences of tRNA genes , 2004, Nucleic Acids Res..

[17]  G. Hannon,et al.  The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis , 2012, Nature.

[18]  Dieter Söll,et al.  Trna: Structure, Biosynthesis, and Function , 1995 .

[19]  Manolis Maragkakis,et al.  Mitochondrial protein BmPAPI modulates the length of mature piRNAs , 2013, RNA.

[20]  D. Haussecker,et al.  Human tRNA-derived small RNAs in the global regulation of RNA silencing. , 2010, RNA.

[21]  S. Le,et al.  Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid , 2009, Nucleic acids research.

[22]  Canan Kuscu,et al.  Biogenesis and Function of Transfer RNA-Related Fragments (tRFs). , 2016, Trends in biochemical sciences.

[23]  Zhiping Weng,et al.  An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. , 2013, Molecular cell.

[24]  S. Eddy,et al.  tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. , 1997, Nucleic acids research.

[25]  Manolis Kellis,et al.  Discrete Small RNA-Generating Loci as Master Regulators of Transposon Activity in Drosophila , 2007, Cell.

[26]  David I. K. Martin,et al.  5′ tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction , 2013, BMC Genomics.

[27]  C. Bracken,et al.  Assessing the gene regulatory properties of Argonaute-bound small RNAs of diverse genomic origin , 2014, Nucleic acids research.

[28]  H. Okano,et al.  Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset, a model primate , 2014, RNA.

[29]  Steven P Gygi,et al.  Angiogenin-induced tRNA fragments inhibit translation initiation. , 2011, Molecular cell.

[30]  Phillipe Loher,et al.  Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers , 2015, Proceedings of the National Academy of Sciences.

[31]  E. Phizicky,et al.  tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis. , 2003, Genes & development.

[32]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[33]  Maike A. Laussmann,et al.  RNA Clamping by Vasa Assembles a piRNA Amplifier Complex on Transposon Transcripts , 2014, Cell.

[34]  J. Jackman,et al.  tRNA 5′-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea , 2010, Nucleic acids research.

[35]  A. Cayota,et al.  A particular set of small non-coding RNAs is bound to the distinctive Argonaute protein of Trypanosoma cruzi: insights from RNA-interference deficient organisms. , 2014, Gene.

[36]  G. Hutvagner,et al.  Transfer RNA‐derived fragments: origins, processing, and functions , 2011, Wiley interdisciplinary reviews. RNA.

[37]  Megumi Shigematsu,et al.  5′-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5′-halves , 2017, RNA.

[38]  Jordan Anaya,et al.  Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets , 2014, BMC Biology.

[39]  J. Lieberman,et al.  G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments , 2014, Proceedings of the National Academy of Sciences.

[40]  P. Ivanov,et al.  YB-1 regulates tiRNA-induced Stress Granule formation but not translational repression , 2016, Nucleic acids research.

[41]  P. Alexiou,et al.  Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway , 2017, RNA.

[42]  Yuan Chang,et al.  Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs , 2012, Nucleic acids research.

[43]  Taishin Kin,et al.  Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells , 2008, Nature.

[44]  R. Sachidanandam,et al.  A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo. , 2010, Genes & development.

[45]  H. Ueda,et al.  The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated , 2007, Nature Structural &Molecular Biology.

[46]  T. Schüpbach,et al.  zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. , 2007, Developmental cell.

[47]  M. Siomi,et al.  Respective functions of two distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells. , 2015, Cell reports.

[48]  E. Phizicky,et al.  tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[49]  F. Tuorto,et al.  RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis , 2012, Nature Structural &Molecular Biology.

[50]  Y. Motorin,et al.  Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme. , 1999, RNA.

[51]  Andrey Grigoriev,et al.  Age-driven modulation of tRNA-derived fragments in Drosophila and their potential targets , 2015, Biology Direct.

[52]  Y. Kirino,et al.  Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method , 2016, Nature Protocols.

[53]  Yi Tie,et al.  Stress induces tRNA cleavage by angiogenin in mammalian cells , 2009, FEBS letters.

[54]  Megumi Shigematsu,et al.  tRNA-Derived Short Non-coding RNA as Interacting Partners of Argonaute Proteins , 2015, Gene regulation and systems biology.

[55]  Markus Glatzel,et al.  CLP1 links tRNA metabolism to progressive motor-neuron loss , 2013, Nature.

[56]  Yi Jing,et al.  Dissecting tRNA-derived fragment complexities using personalized transcriptomes reveals novel fragment classes and unexpected dependencies , 2015, Oncotarget.

[57]  Robert Blelloch,et al.  Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. , 2008, Genes & development.

[58]  Pavel Ivanov,et al.  Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly* , 2010, The Journal of Biological Chemistry.

[59]  Eugene Berezikov,et al.  A Role for Piwi and piRNAs in Germ Cell Maintenance and Transposon Silencing in Zebrafish , 2007, Cell.

[60]  Jernej Ule,et al.  Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders , 2014, The EMBO journal.

[61]  Yongfeng Jin,et al.  RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori , 2013, BMC Genomics.

[62]  A. Aravin,et al.  PIWI-interacting small RNAs: the vanguard of genome defence , 2011, Nature Reviews Molecular Cell Biology.

[63]  Kuniaki Saito,et al.  Structure and function of Zucchini endoribonuclease in piRNA biogenesis , 2012, Nature.

[64]  Pavel Ivanov,et al.  tRNA fragments in human health and disease , 2014, FEBS letters.

[65]  Kuniaki Saito,et al.  Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends. , 2007, Genes & development.

[66]  A. Hopper,et al.  tRNA biology charges to the front. , 2010, Genes & development.

[67]  Manolis Maragkakis,et al.  Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm , 2016, Nature.

[68]  T. Speed,et al.  A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus. , 2012, Molecular cell.