Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, which afflicts more than 200 million people yearly in tropical regions of South America, Asia and Africa. A promising approach to the control of this and many other diseases involves the application of our understanding of small non-coding RNA function to the design of safe and effective means of treatment. In a previous study, we identified five conserved miRNAs from the adult stage of Schistosoma japonicum. Here, we applied Illumina Solexa high-throughput sequencing methods (deep sequencing) to investigate the small RNAs expressed in S. japonicum schistosomulum (3 weeks post-infection). This has allowed us to examine over four million sequence reads including both frequently and infrequently represented members of the RNA population. Thus we have identified 20 conserved miRNA families that have orthologs in well-studied model organisms and 16 miRNA that appear to be specific to Schistosoma. We have also observed minor amounts of heterogeneity in both 3′ and 5′ terminal positions of some miRNA as well as RNA fragments resulting from the processing of miRNA precursor. An investigation of the genomic arrangement of the 36 identified miRNA revealed that seven were tightly linked in two clusters. We also identified members of the small RNA population whose structure indicates that they are part of an endogenously derived RNA silencing pathway, as evidenced by their extensive complementarities with retrotransposon and retrovirus-related Pol polyprotein from transposon.
References
[1]
Ivey KN, Muth A, Arnold J, King FW, Yeh RF, et al. (2008) MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2: 219–229. doi: 10.1016/j.stem.2008.01.016
[2]
Kim VN (2005) Small RNAs: classification, biogenesis, and function. Mol Cells 19: 1–15.
[3]
Malone CD, Hannon GJ (2009) Small RNAs as guardians of the genome. Cell 136: 656–668. doi: 10.1016/j.cell.2009.01.045
[4]
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10: 94–108. doi: 10.1038/nrg2504
[5]
Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432: 231–235. doi: 10.1038/nature03049
[6]
Lee Y, Ahn C, Han J, Choi H, Kim J, et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425: 415–419. doi: 10.1038/nature01957
[7]
Ketting RF, Fischer SEJ, Bernstein E, Sijen T, Hannon GJ, et al. (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C-elegans. Genes & Development 15: 2654–2659. doi: 10.1101/gad.927801
[8]
Schwarz DS, Hutvagner G, Du T, Xu ZS, Aronin N, et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199–208. doi: 10.1016/S0092-8674(03)00759-1
Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes & Development 19: 517–529. doi: 10.1101/gad.1284105
[11]
Farazi TA, Juranek SA, Tuschl T (2008) The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 135: 1201–1214. doi: 10.1242/dev.005629
[12]
Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nature Reviews Molecular Cell Biology 10: 126–139. doi: 10.1038/nrm2632
[13]
Vagin VV, Sigova A, Li CJ, Seitz H, Gvozdev V, et al. (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313: 320–324. doi: 10.1126/science.1129333
[14]
Zhou Y, Zheng HJ, Chen YY, Zhang L, Wang K, et al. (2009) The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature 460: 345–U356. doi: 10.1038/nature08140
[15]
Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, et al. (2009) The genome of the blood fluke Schistosoma mansoni. Nature 460: 352–U365. doi: 10.1038/nature08160
[16]
Xue X, Sun J, Zhang Q, Wang Z, Huang Y, et al. (2008) Identification and characterization of novel microRNAs from Schistosoma japonicum. PLoS ONE 3: e4034. doi: 10.1371/journal.pone.0004034
[17]
Morin RD, O'Connor MD, Griffith M, Kuchenbauer F, Delaney A, et al. (2008) Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Research 18: 610–621. doi: 10.1101/gr.7179508
[18]
Glazov EA, Cottee PA, Barris WC, Moore RJ, Dalrymple BP, et al. (2008) A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. Genome Research 18: 957–964. doi: 10.1101/gr.074740.107
[19]
Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43: 110–117. doi: 10.1016/j.ymeth.2007.05.002
[20]
Lopez R, Silventoinen V, Robinson S, Kibria A, Gish W (2003) WU-Blast2 server at the European Bioinformatics Institute. Nucleic Acids Res 31: 3795–3798. doi: 10.1093/nar/gkg573
[21]
Gardner PP, Daub J, Tate JG, Nawrocki EP, Kolbe DL, et al. (2009) Rfam: updates to the RNA families database. Nucleic Acids Research 37: D136–D140. doi: 10.1093/nar/gkn766
[22]
Hofacker IL (2003) Vienna RNA secondary structure server. Nucleic Acids Research 31: 3429–3431. doi: 10.1093/nar/gkg599
[23]
Gruber AR, Lorenz R, Bernhart SH, Neuboock R, Hofacker IL (2008) The Vienna RNA Websuite. Nucleic Acids Research 36: W70–W74. doi: 10.1093/nar/gkn188
[24]
Ruby JG, Jan C, Player C, Axtell MJ, Lee W, et al. (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C-elegans. Cell 127: 1193–1207. doi: 10.1016/j.cell.2006.10.040
[25]
Ambros V, Lee RC, Lavanway A, Williams PT, Jewell D (2003) MicroRNAs and other tiny endogenous RNAs in C-elegans. Current Biology 13: 807–818. doi: 10.1016/S0960-9822(03)00287-2
[26]
Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294: 858–862. doi: 10.1126/science.1065062
[27]
Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research 33: 9. doi: 10.1093/nar/gni178
[28]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262
[29]
Horwich MD, Li CJ, Matranga C, Vagin V, Farley G, et al. (2007) The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Current Biology 17: 1265–1272. doi: 10.1016/j.cub.2007.06.030
[30]
Alefelder S, Patel BK, Eckstein F (1998) Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Research 26: 4983–4988. doi: 10.1093/nar/26.21.4983
[31]
Ruby JG, Stark A, Johnston WK, Kellis M, Bartel DP, et al. (2007) Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. Genome Research 17: 1850–1864. doi: 10.1101/gr.6597907
[32]
miRBase.[http://microrna.sanger.ac.uk/registry/].
[33]
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Research 36: D154–D158. doi: 10.1093/nar/gkm952
[34]
Yao YY, Guo GG, Ni ZF, Sunkar R, Du JK, et al. (2007) Cloning and characterization of microRNAs from wheat (Triticum aestivum L.). Genome Biology 8: 13. doi: 10.1186/gb-2007-8-6-r96
[35]
Hertel J, Lindemeyer M, Missal K, Fried C, Tanzer A, et al. (2006) The expansion of the metazoan microRNA repertoire. Bmc Genomics 7:
[36]
Bar M, Wyman SK, Fritz BR, Qi JL, Garg KS, et al. (2008) MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries. Stem Cells 26: 2496–2505. doi: 10.1634/stemcells.2008-0356
[37]
Liu G, Min H, Yue S, Chen CZ (2008) Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development. PLoS ONE 3: e3592. doi: 10.1371/journal.pone.0003592
[38]
Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes & Development 20: 3407–3425. doi: 10.1101/gad.1476406
[39]
Reddy AM, Zheng Y, Jagadeeswaran G, Macmil SL, Graham WB, et al. (2009) Cloning, characterization and expression analysis of porcine microRNAs. BMC Genomics 10: 65. doi: 10.1186/1471-2164-10-65
[40]
Palakodeti D, Smielewska M, Graveley BR (2006) MicroRNAs from the Planarian Schmidtea mediterranea: A model system for stem cell biology. Rna-a Publication of the Rna Society 12: 1640–1649. doi: 10.1261/rna.117206
[41]
Lu C, Tej SS, Luo SJ, Haudenschild CD, Meyers BC, et al. (2005) Elucidation of the small RNA component of the transcriptome. Science 309: 1567–1569. doi: 10.1126/science.1114112
[42]
Barad O, Meiri E, Avniel A, Aharonov R, Barzilai A, et al. (2004) MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res 14: 2486–2494. doi: 10.1101/gr.2845604
[43]
Sempere LF, Freemantle S, Pitha-Rowe I, Moss E, Dmitrovsky E, et al. (2004) Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol 5: R13. doi: 10.1186/gb-2004-5-3-r13
Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, et al. (2003) A uniform system for microRNA annotation. Rna 9: 277–279. doi: 10.1261/rna.2183803
[46]
Okamura K, Balla S, Martin R, Liu N, Lai EC (2008) Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat Struct Mol Biol 15: 581–590. doi: 10.1038/nsmb.1438
[47]
Okamura K, Chung WJ, Ruby JG, Guo HL, Bartel DP, et al. (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453: 803–U808. doi: 10.1038/nature07015
[48]
Chung WJ, Okamura K, Martin R, Lai EC (2008) Endogenous RNA interference provides a somatic defense against Drosophila transposons. Curr Biol 18: 795–802. doi: 10.1016/j.cub.2008.05.006
[49]
Czech B, Malone CD, Zhou R, Stark A, Schlingeheyde C, et al. (2008) An endogenous small interfering RNA pathway in Drosophila. Nature 453: 798–802. doi: 10.1038/nature07007
[50]
Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, et al. (2008) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320: 1077–1081. doi: 10.1126/science.1157396
[51]
Kawamura Y, Saito K, Kin T, Ono Y, Asai K, et al. (2008) Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453: 793–U795. doi: 10.1038/nature06938
[52]
Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453: 534–538. doi: 10.1038/nature06904
[53]
Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453: 539–U539. doi: 10.1038/nature06908
[54]
Golden DE, Gerbasi VR, Sontheimer EJ (2008) An inside job for siRNAs. Molecular Cell 31: 309–312. doi: 10.1016/j.molcel.2008.07.008
[55]
Copeland CC, Marz M, Rose D, Hertel J, Brindley PJ, et al. (2009) Homology-based annotation of non-coding RNAs in the genomes of Schistosoma mansoni and Schistosoma japonicum. BMC Genomics 10: 464. doi: 10.1186/1471-2164-10-464
[56]
Ambros V (2004) The functions of animal microRNAs. Nature 431: 350–355. doi: 10.1038/nature02871
[57]
Leaman D, Chen PY, Fak J, Yalcin A, Pearce M, et al. (2005) Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121: 1097–1108. doi: 10.1016/j.cell.2005.04.016
[58]
Enright AJ, John B, Gaul U, Tuschl T, Sander C, et al. (2003) MicroRNA targets in Drosophila. Genome Biol 5: R1. doi: 10.1186/gb-2003-5-1-r1
[59]
Yu X, Zhou Q, Li SC, Luo Q, Cai Y, et al. (2008) The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages. PLoS ONE 3: e2997. doi: 10.1371/journal.pone.0002997
[60]
Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters. Science 322: 1845–1848. doi: 10.1126/science.1162228
[61]
Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, et al. (2008) Divergent Transcription from Active Promoters. Science 322: 1849–1851. doi: 10.1126/science.1162253
[62]
Buratowski S (2008) Transcription. Gene expression–where to start? Science 322: 1804–1805. doi: 10.1126/science.1168805
[63]
Okamura K, Lai EC (2008) Endogenous small interfering RNAs in animals. Nature Reviews Molecular Cell Biology 9: 673–678. doi: 10.1038/nrm2479
