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Small RNA Research

Why to Study piRNA?

 

Introduction

Piwi-interacting RNAs (piRNAs) are the largest class of single stranded, small non-coding RNAs of about 26-32 nucleotides in length. piRNAs interact with the Piwi (P-element Induced Wimpy Testis) subfamily of Argonaute proteins. The Piwi subfamily comprises Piwi, Aubergine and AGO3 in flies, MILI, MIWI and MIWI2 in mice, and HILI, HIWI1, HIWI2 and HIWI3 in humans [1]. Individual piRNAs are very diverse and are poorly conserved even between closely related species. In contrast to several hundred microRNA species, tens of thousands of unique piRNA sequences are known in human, mouse and rat [2].  piRNAs are strikingly different from microRNAs in their length, expression pattern, genomic organization and biogenesis.

Biogenesis of piRNAs

Unlike miRNAs and siRNAs, piRNAs are not generated from dsRNA precursors by Dicer. Rather, piRNAs are produced from a primary transcript that traverses an entire piRNA cluster and is subsequently processed into mature piRNAs. In Drosophila, the first 10 nucleotides of piRNAs bound to Aub or Piwi, which typically begin with uridine, are often complementary to the first 10 nucleotides of piRNAs bound to Ago3, which usually contain an adenosine at position 10. The observations led to the proposal of the "ping-pong" model, in which new piRNAs are generated by amplification mediated by this complementarity [3].  piRNA biogenesis also spreads along the precursor sequence in a Zucchini-dependent and phased manner from the site of initial piRNA formation. The mechanism explains the extraordinary sequence diversity of piRNAs [7,8].

pi-1

Fig.1 The model for piRNA biogenesis. In germline cells, the piRNA precursor transcribed from a piRNA cluster is cleaved successively by Zucchini (Zuc) endonuclease activity as phased by Piwi loading. The RNA intermediates having a 5’U are preferentially loaded into Aub, trimmed as necessary, and 2’O-methylated by Hen1 to produce the mature primary piRNA. The primary piRNA then guides to the transposon transcript or secondary piRNA precursor to initiate the production of secondary piRNA by loading to Ago3. The secondary piRNA in turn triggers the next cycle of primary piRNA pathway, reciprocating the “Ping-pong cycles” that amplify piRNAs and consume transposon transcripts.

Function of piRNAs

The mammalian piRNAs can be divided into two classes, pre-pachytene and pachytene piRNAs, depending on the stage of meiosis at which they are expressed in developing spermatocytes [2]. They may have distinct functions according to their sequence features[1].

pi-2

Fig 2. piRNAs in mouse, associated with Piwi proteins, such as Mili and Miwi, can be divided into two classes with divergent functions [4].

• Germline development and spermatogenesis: Complete loss of Miwi and Mili causes meiotic arrest during spermatogenesis, leading to seminiferous tubules devoid of sperm.
• Transposon silencing and preservation of genomic integrity: Mili-and Miwi-2 null mice have increased activity of retrotransposons, suggesting that piRNAs protect the germline genome from deleterious transposon insertions [6]. A large number of piRNAs are mapped to the transposon-containing genomic loci. Transposon transcription is repressed by the piRNA pathway. The slicer activity of Aub and Ago3 consumes transposon transcripts at post-transcriptional level.
• Epigenetic regulation: PIWI–piRNA pathway is involved in epigenetic regulation through histone modification, DNA methylation and heterochromatin assembly.
• Regulation of translation and mRNA turn-over: Miwi and piRNAs associate with the cap binding complex and modulate mRNA stability during the production of proteins required for spermatogenesis [5]. 3’UTRs of a broad set of protein-coding mRNAs produce piRNAs [9].
• In somatic cells, maternal PIWI proteins are essential for the maintenance of chromatin structure and cell cycle progression during early embryogenesis.
• In human, PIWI proteins and the piRNA pathway may be linked to cancers [10].

Perspective

Although piRNAs have been discovered in large numbers in multiple species, we still do not know how a new piRNA response is initiated, and the exact function of piRNAs in development remains elusive. There are many open questions in the piRNA field ripe for further study. Due to the large number of piRNAs, characterization of individual piRNAs is more challenging than that of single miRNAs. However, the refinement of microarray and high-throughput sequencing techniques will enable researchers to learn much more about the biological roles of individual piRNAs in the near future. Additionally, piRNAs have been explored as small RNA cancer biomarkers or tissue signatures.

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References 
1. Ghildiyal M., Zamore PD. (2009). Small silencing RNAs: an expanding universe. Nat Rev Genet. 10(2):94-108. 2. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., et al. (2006). A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203-207. 
3. Haifan Lin. (2007). piRNAs in the Germ Line. Science 316, 397. 
4. Molecular Biology Select. (2006). Cell. 126:223-225. 
5. Grivna, S.T., Beyret, E., Wang, Z., and Lin, H. (2006). A novel class of small RNAs in mouse spermatogenic cells. Genes Dev. 20, 1709-714. 
6. Stefani G, Slack FJ. (2008). Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol. 9(3):219-30. 
7. Mohn, F., Handler, D. and J. Brennecke (2015).  Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis. Science 348: 812-7.
8. Han, B. W., Wang, W., Li, C., Weng, Z., P.D. Zamore (2015) Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science 348:817-21.
9. Robine N, Lau NC, Balla S, et al. (2009) A broadly conserved pathway generates 3'UTR-directed primary piRNAs. Curr Biol. 2009; 19:2066–76.
10. Ross RJ, Weiner MM, Lin H. (2014) PIWI proteins and PIWI-interacting RNAs in the soma. Nature.505:353–9.

 

 

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