Retrotransposon Silencing During Embryogenesis: Dicer Cuts in LINE

Fossilised mobile genetic elements, including Long Interspersed Element-1 (LINE-1 or L1) retrotransposons, comprise at least two-thirds of the human genome [1]. Their molecular history is reminiscent of speciation and natural selection, where, as noted by Carl Sagan, ‘‘Extinction is the rule. Survival is the exception’’ [2]. Broadly, the life cycle of a retrotransposon begins with innovation to evade host genome surveillance, followed by ‘‘copyand-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 [3]. Even more impressively, approximately 500,000 copies of a single retrotransposon superfamily, Gypsy, occupy nearly half of the maize genome [4]. 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 DNAprimed 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 [5–8]. A plausible model of rasiRNA biogenesis involves bidirectional transcription of opposed retrotransposon promoters [9,10], 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 RNAinduced silencing complex (RISC) that, in turn, produces RNAi against retrotransposon transcripts [11]. The suppressive influence of rasiRNAs, in concert with other pathways, may explain why retrotransposition is more common during embryogenesis than in gametes [12,13]. Importantly, although rasiRNAs have been found in stem cells and soma, their capacity to suppress retrotransposition during development is relatively unexplored [14–16]. In this issue of PLOS Genetics, Ciaudo et al. [17] 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 59UTR [15], 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 DCR controls, based on SYBR-Green qPCR targeting the L1-Tf 59UTR. 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 Dicerand AGO2-independent small RNAs were found to ‘‘paint’’ the L1-Tf 59UTR. 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 [18], as well as evidence of elevated L1-Tf expression in DGCR8 mESCs [19]. 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