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The Potential Regulation of L1 Mobility by RNA Interference

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RNAi silencing of transposable elementsL1 retrotransposition: hazardsand constraintsL1 RNA is a logical target for limiting L1 mobilityHow might RNAi silence L1s?Establishing a functional link betweenRNAi and L1 retrotranspositionAlternative silencing pathways mediatedby dsRNAConclusionAcknowledgmentsREFERENCESHindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2006, Article ID 32713, Pages 1–8DOI 10.1155/JBB/2006/32713Review ArticleThe Potential Regulation of L1 Mobility by RNA InterferenceShane R. Horman,1Petr Svoboda,2and Eline T. Luning Prak11Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia,PA 19104-6055, USA2Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, SwitzerlandReceived 6 August 2005; Revised 12 December 2005; Accepted 20 December 2005The hypothesis that RNA interference constrains L1 mobility seems inherently reasonable: L 1 mobility can be dangerous andL1 RNA, the presumed target of RNAi, serves as a critical retrotransposition intermediate. Despite its plausibility, proof for thishypothesis has been difficult to obtain. Studies attempting to link the L1 retrotransposition frequency to alterations in RNAiactivity have been hampered by the long times required to measure retrotransposition frequency, the pleiotropic and toxic effectsof altering RNAi over similar time periods, and the possibility that other cellular machinery may contribute to the regulationof L1s. Another problem is that the commonly used L1 reporter cassette may serve as a substrate for RNAi. Here we review theL1-RNAi hypothesis and descr ibe a genetic assay with a modified reporter cassette that detects approximately 4 times more L1insertions than the conventional retrotransposition assay.Copyright © 2006 Shane R. Horman et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.RNAi SILENCING OF TRANSPOSABLE ELEMENTSRNAi is an evolutionarily conserved process of sequence-specific posttr a nscriptional gene silencing (reviewed in [1]).Double-stranded RNA (dsRNA) is cleaved by the ribonu-clease DICER into small interfering RNA species (siRNAs).SiRNA molecules, in turn, target complementary RNA se-quences for destruction (reviewed in [2]). RNAi is postu-lated to play a role in the silencing of transposable elementsand viruses that produce dsRNA [3, 4]. One line of evidencelinking RNAi to repressed transposition comes from the ne-matode, Celegans[5, 6]. Tc1 elements, a class of DNA tr a ns-posons, mobilize in somatic cells, but are silenced in the germline of Celegans.AnumberofmutantCelegansstrains thathave lost this silencing have also lost the ability to executeRNAi (though there were also RNAi mutants that lacked thistransposon mobilization phenotype) [5]. The identificationof specific genes, which when mutated show activation ofgermline transposition, indicates that an active transposon-silencing process exists in the germline [5, 6]. Another lineof evidence linking RNAi (or a mechanism similar to RNAi)to the regulation of transposable elements involves the I-factor in Drosophila. Mobilization of the I-factor (an L1-likenon-LTR retrotransposon) is regulated at least in part bya homology-dependent silencing mechanism in the femalegermline [7, 8]. This silencing mechanism has been linked toa series of molecules that are implicated in the RNAi pathway,including the Argonaute protein PIWI [9, 10].By analogy, perhaps a sequence-dependent process ofmobile element silencing, such as RNAi, is used to regulateL1 mobility. As with the above-mentioned examples, the reg-ulation of L1 mobility may be particularly relevant in thegermline and in embryos. Mobility in the germline or inembryos could result in inheritance of the new insertion.These sites are a lso where L1s are believed to b e most active[11–14]. Other mechanisms for recognizing and respondingto dsRNA, such as RNase L and PKR-mediated responses,can cause apoptosis. While apoptosis seems like a reason-able strategy for dealing with a wayward somatic cell, in thegermline or early embr yo, apoptosis could be detrimental tothe fitness of the organism [14, 15]. Here we explore the the-sis that the mobility of human L1s is regulated by RNAi.L1 RETROTRANSPOSITION: HAZARDSAND CONSTRAINTSThe human genome contains roughly half a million long in-terspersed elements (L1s) that collectively account for 17%ofitsmass[16]. Most new L1 inser tions are “dead on arrival”due to 5truncation and nearly all but perhaps 60–100 L1 se-quences in the human genome are inactive due to truncation,inversion, or mutation [17].2 Journal of Biomedicine and BiotechnologyAs discussed elsewhere in this issue, retrotranspositioncan be hazardous because L1s can insert into genes, altergene expression, shuffle exons, transduce 3flanking sequen-ces, mobilize Alu elements, and their replicative mobilizationadds significant DNA mass to the genome [18–24]. L1 inser-tions and recombination events involving genomic L1 andAlu insertions have been reported in a number of genetic dis-orders (reviewed in [25]). Although it is possible that somefunctions of L1 are beneficial to mammals (a most interest-ing recent demonstration involves the potential role of L1sas diversity generators in the CNS, [26]), most germline L1insertions are likely to be neut ral or negatively selected. Neg-ative selection of L1s is suggested by the higher frequenciesof full-length human L1 insertions on the sex chromosomesthan the autosomes (the former not being as able as the latterto undergo purifying selec tion) and by the dominance andlimited periods of activity of single L1 subfamilies in someprimate lineages [27, 28].L1 mobility in mammals appears to be actively con-strained. An indirect line of evidence for this constraint isthat different cell types exhibit different rates of retrotrans-position, ranging from 30% or higher in some transformedcell lines to fewer than one per million cells. In the mouse,the rate of germline retrotransposition events using an L1-EGFP transgene is approximately one event in 100 offspring[11, 13]. Analysis of L1 transcription, protein productionand retrotransposition, reveals different levels of L1 activityin different cell types, with highest levels of activity noted ingerm cells,


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