Citation: Faulkner GJ (2013) Retrotransposon Silencing During Embryogenesis: Dicer Cuts in LINE. PLoS Genet 9(11): e1003944. doi:10.1371/journal.pgen.1003944
Editor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom
Published: November 7, 2013
Copyright: © 2013 Geoffrey J. Faulkner. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The author is supported by an Australian NHMRC Career Development Fellowship (GNT1045237), NHMRC Project Grants GNT1042449, GNT1045991, and GNT1052303, and the European Union's Seventh Framework Programme (FP7/2007–2013) under grant agreement No. 259743 underpinning the MODHEP consortium. The funders had no role in the preparation of the manuscript.
Competing interests: The author has declared that no competing interests exist.
Fossilised mobile genetic elements, including Long Interspersed Element-1 (LINE-1 or L1) retrotransposons, comprise at least two-thirds of the human genome . Their molecular history is reminiscent of speciation and natural selection, where, as noted by Carl Sagan, “Extinction is the rule. Survival is the exception” . Broadly, the life cycle of a retrotransposon begins with innovation to evade host genome surveillance, followed by “copy-and-paste” retrotransposition and, finally, quiescence as a result of host defence adaptation. Before being tamed, a new or newly reactivated retrotransposon can undergo massive copy number amplification. For instance, more than one million copies of the primate-specific Short Interspersed Element (SINE) Alu comprise 11% of the human genome . Even more impressively, approximately 500,000 copies of a single retrotransposon superfamily, Gypsy, occupy nearly half of the maize genome . Thus, retrotransposons can overrun a genome within a brief evolutionary period, making their suppression a high host priority.
Retrotransposition requires transcription of an RNA template for DNA-primed reverse transcription. Several cellular defence mechanisms have evolved to hinder this process, including: 1) promoter methylation and heterochromatinisation, 2) degradation of retrotransposon transcripts via RNA interference (RNAi), and 3) host factor prevention or destabilisation of reverse transcription. To describe in detail just one of a myriad of specific inhibitory pathways, repeat associated small interfering RNAs (rasiRNAs) are present in plant, worm, fly, fish, and mouse gametes and, therefore, represent a highly conserved defence against germ line retrotransposition –. A plausible model of rasiRNA biogenesis involves bidirectional transcription of opposed retrotransposon promoters , , resulting in the formation of double-stranded RNAs (Figure 1). These are cleaved by Dicer (DCR) and then assembled with Argonaute (AGO) and other proteins into the RNA-induced silencing complex (RISC) that, in turn, produces RNAi against retrotransposon transcripts . The suppressive influence of rasiRNAs, in concert with other pathways, may explain why retrotransposition is more common during embryogenesis than in gametes , . Importantly, although rasiRNAs have been found in stem cells and soma, their capacity to suppress retrotransposition during development is relatively unexplored –.
Mouse LINE-1s are comprised of two ORFs flanked by 5′ and 3′UTRs. Several monomers in the 5′UTR provide promoter activity. Following the LINE-1 expression and copy number variation data of Ciaudo et al., bidirectional transcription of the 5′UTR generates sense and antisense LINE-1 RNAs. The Drosha-DGCR8 Microprocessor cleaves these precursors into pre-miRNAs, which are processed into miRNAs by Dicer, but may not be loaded into the RISC complex. By contrast, double-stranded RNAs potentially formed by sense/antisense pairing of LINE-1 RNAs are also cleaved by Dicer but here generate rasiRNAs, loaded into the RISC complex, which degrade canonical LINE-1 mRNAs. Dicer also appears to mediate LINE-1 promoter methylation (not shown).
In this issue of PLOS Genetics, Ciaudo et al.  describe rasiRNA-mediated suppression of LINE-1 activity in mouse embryonic stem cells (mESCs). Focusing on the L1-Tf subfamily, where they previously described an unusual rasiRNA signature mapping to the 5′UTR , Ciaudo et al. observed that knock-out of Dicer markedly decreases L1-Tf promoter methylation and increases L1-Tf transcription, translation, and copy number in cultured mESCs. In particular, DCR−/− mESCs accumulate a remarkable 860 L1-Tf copies (greater than five megabases of genomic DNA) per cell over 20 passages, versus 255 copies per cell in DCRFlx/Flx controls, based on SYBR-Green qPCR targeting the L1-Tf 5′UTR. High-throughput small RNA sequencing then confirmed that DCR−/− mESCs were depleted of approximately 22 nt molecules found in wild-type mESCs, immunoprecipitated with AGO2 and aligned to L1-Tf, and therefore resembling rasiRNAs. Hence, LINE-1 activation in DCR−/− mESCs coincides with rasiRNA depletion and is also possibly influenced by ablation of Dicer-mediated LINE-1 promoter methylation.
Intriguingly, a second class of Dicer- and AGO2-independent small RNAs were found to “paint” the L1-Tf 5′UTR. Again, assessing L1-Tf transcription and copy number, Ciaudo et al. found that deletion of XRN2 and DGCR8, respective members of the RNA surveillance and Drosha-DGCR8 Microprocessor pathways, led to increased L1-Tf transcription but not copy number amplification. These observations agree with other recent reports of small RNAs immunoprecipitated with DGCR8 and enriched for LINE-1 sequences , as well as evidence of elevated L1-Tf expression in DGCR8−/− mESCs . As a final experiment, Ciaudo et al. complemented DCR−/− mESCs with human Dicer and found that these cells recapitulated wild-type mESC LINE-1 suppression and differentiated normally, unlike DCR−/− mESCs.
Evidence for a reciprocal relationship between rasiRNA depletion and LINE-1 activation significantly advances our understanding of RNAi-mediated control of retrotransposition during mammalian embryogenesis. These data are also important because they address a longstanding question of why rasiRNAs cannot be consistently detected in mammalian somatic cells: small RNAs generated by RNA surveillance and the Microprocessor may cleave the same pool of precursor LINE-1 mRNAs processed by Dicer and obscure rasiRNA detection (Figure 1). As Ciaudo et al. note, it is possible that insertional mutagenesis caused by LINE-1 contributes to the reported differentiation defects for DCR−/− mESCs , though it is unclear why lesser but still substantial LINE-1 activity is tolerated by wild-type mESCs. Interestingly, experiments using engineered LINE-1 reporters have shown elsewhere ,  that mutation of Dicer or the Microprocessor increases LINE-1 mobilisation in cancer cells, with the latter result at odds with data generated here from mESCs. Future advances in high-throughput sequencing and single cell genomics should enable characterisation of endogenous LINE-1 mobilisation events in stem cells and further delineate the multifaceted roles of Dicer and other factors in LINE-1 inhibition.
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