Abstract
Piwi-interacting RNAs (piRNA) are fully modified by 2′-O-methylation at their 3′-termini. This terminal methylation is required to prevent 3′-nucleotide addition, which serves as a tag for destabilization. In this chapter, we describe biochemical and mass spectrometric analyses of 2′-O-methylation at 3′-termini of piRNAs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Juliano C, Wang J, Lin H (2011) Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms. Annu Rev Genet 45:447–469
Siomi MC, Sato K, Pezic D, Aravin AA (2011) PIWI-interacting small RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol 12:246–258
Pillai RS, Chuma S (2012) piRNAs and their involvement in male germline development in mice. Dev Growth Differ 54:78–92
Kirino Y, Mourelatos Z (2007) Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini. Nat Struct Mol Biol 14:347–348
Ohara T, Sakaguchi Y, Suzuki T, Ueda H, Miyauchi K (2007) The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated. Nat Struct Mol Biol 14:349–350
Horwich MD, Li C, Matranga C, Vagin V, Farley G, Wang P, Zamore PD (2007) The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr Biol 17:1265–1272
Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RH, Hannon GJ, Draper BW, Ketting RF (2007) A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129:69–82
Zhou X, Zuo Z, Zhou F, Zhao W, Sakaguchi Y, Suzuki T, Cheng H, Zhou R (2010) Profiling sex-specific piRNAs in zebrafish. Genetics 186:1175–1185
Saito K, Sakaguchi Y, Suzuki T, Suzuki T, Siomi H, Siomi MC (2007) Pimet, the Drosophila homolog of HEN1, mediates 2′-O-methylation of Piwi-interacting RNAs at their 3′ ends. Genes Dev 21:1603–1608
Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R, Chen X (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307:932–935
Yang Z, Ebright YW, Yu B, Chen X (2006) HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 34:667–675
Huang Y, Ji L, Huang Q, Vassylyev DG, Chen X, Ma JB (2009) Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461:823–827
Zhao Y, Mo B, Chen X (2012) Mechanisms that impact microRNA stability in plants. RNA Biol 9:1218–1223
Huang RH (2012) Unique 2′-O-methylation by Hen1 in eukaryotic RNA interference and bacterial RNA repair. Biochemistry 51:4087–4095
Kirino Y, Mourelatos Z (2007) The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs. RNA 13:1397–1401
Kamminga LM, Luteijn MJ, den Broeder MJ, Redl S, Kaaij LJ, Roovers EF, Ladurner P, Berezikov E, Ketting RF (2010) Hen1 is required for oocyte development and piRNA stability in zebrafish. EMBO J 29:3688–3700
Billi AC, Alessi AF, Khivansara V, Han T, Freeberg M, Mitani S, Kim JK (2012) The Caenorhabditis elegans HEN1 ortholog, HENN-1, methylates and stabilizes select subclasses of germline small RNAs. PLoS Genet 8:e1002617
Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, Lai EC (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453:803–806
Kurth HM, Mochizuki K (2009) 2′-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena. RNA 15:675–685
Kawaoka S, Izumi N, Katsuma S, Tomari Y (2011) 3′ end formation of PIWI-interacting RNAs in vitro. Mol Cell 43:1015–1022
Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, Zamore PD (2010) Target RNA-directed trimming and tailing of small silencing RNAs. Science 328:1534–1539
Suzuki T, Ikeuchi Y, Noma A, Sakaguchi Y (2007) Mass spectrometric identification and characterization of RNA-modifying enzymes. Methods Enzymol 425:211–229
Kimura S, Ikeuchi Y, Kitahara K, Sakaguchi Y, Suzuki T, Suzuki T (2012) Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity. Nucleic Acids Res 40:4071–4085
Suzuki T, Sakaguchi Y, Suzuki T (2007) Mass spectrometric analysis of 3′-terminal nucleosides of non-coding RNAs. Protoc Exch. doi:10.1038/nprot.2007.1185
Acknowledgments
We sincerely appreciate the Suzuki laboratory members for fruitful discussion on this project. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan (to Takeo S. and Tsutomu S.) and by a grant from the New Energy and Industrial Technology Development Organization (NEDO) (to Tsutomu S.).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Suzuki, T., Miyauchi, K., Sakaguchi, Y., Suzuki, T. (2014). Biochemical and Mass Spectrometric Analysis of 3’-End Methylation of piRNAs. In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-62703-694-8_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-693-1
Online ISBN: 978-1-62703-694-8
eBook Packages: Springer Protocols