- Email: [email protected]
Transposable element small RNAs as regulators of gene expression
Review
Transposable element small RNAs as regulators of gene expression Andrea D. McCue and R. Keith Slotkin Department of Molecular Genetics & Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Transposable elements (TEs) are a source of endogenous small RNAs in animals and plants. These TE-derived small RNAs have been traditionally treated as functionally distinct from gene-regulating small RNAs, such as miRNAs. Two recent reports in Drosophila and Arabidopsis have blurred the lines of this distinction. In both examples, epigenetically and developmentally regulated bursts in TE expression produce gene-regulating small RNAs. In the Drosophila early embryo, maternally deposited TEderived PIWI-interacting small RNAs (piRNAs) play a role in regulating the nanos mRNA through small RNA binding sites in the nanos 30 untranslated region (UTR). In Arabidopsis, when Athila retrotransposons are epigenetically activated, their transcripts are processed into small RNAs, which directly target the 30 UTR of the genic oligouridylate binding protein 1B (UBP1b) mRNA. Based on these two examples, we suggest that other TE-derived small RNAs regulate additional genes and propose that, through small RNAs, the epigenetic status of TEs could widely influence the genic transcriptome. The long-held distinction between gene-regulating and TE-regulating small RNAs The regulation of genes has long been treated as an independent pathway from the regulation of TEs. Even as new examples of genes or genome-wide processes that are regulated by TEs appear in publication almost monthly, current dogma still separates the regulation of the relatively clean single-copy genes from the messy repetitive genome context in which they reside. One such long-standing functional separation between TE and gene has been in the classes of small RNAs they produce. The literature describing small RNA biology has generally categorized distinctly separate small RNA biogenesis pathways for the regulation of repetitive, TE-rich regions of the genome (Box 1) and for the regulation of genes (Box 2) [1–3]. TEs are major producers of endogenous small interfering RNAs (endo-siRNAs) in plants and animals and piRNAs in animals (Table 1). These two classes of small RNAs act to repress TE mRNA accumulation post-transcriptionally (reviewed in [2]) and, in some cases, to induce DNA methylation and repressive histone tail modifications at TE loci to maintain the element in a transcriptionally repressed heterochromatic state [4–7]. TE regulation has long been known to influence proximal neighboring genes by readthrough transcription or ectopic recruitment of silencing Corresponding author: Slotkin, R.K. ([email protected]) Keywords: transposable element; small RNAs; piRNA; siRNA; gene regulation.
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factors (reviewed in [1,8]). However, two recently published examples use fundamentally distinct mechanisms whereby host genes regulated by the TE do not have to be adjacent to or associated with any TE fragment. In these cases, a TE-derived small RNA acts in trans in a similar fashion as a miRNA, post-transcriptionally or translationally repressing target genes with imperfect complementarity. These two cases blur the lines between the wellengrained distinction of gene-regulating and TE-derived small RNAs. Initial inquiries into gene-regulating TE small RNAs The idea that TE-derived small RNAs could regulate genes is not new. Several virus-derived miRNAs were identified in 2004 [9], and it is likely that these miRNAs post-transcriptionally alter host gene expression [10,11]. In 2005, miRNAs were also predicted from mammalian genomic repeat sequences [12], suggesting that, similar to viruses, TE miRNAs could influence genes in a post-transcriptional manner. TEs may also play a role in the evolution of miRNAs. miRNA evolution is postulated to initiate from short inverted duplications of genes, because these inverted duplications could form extended hairpin foldback structures that are acted upon by Dicer proteins, producing endo-siRNAs that have the ability to target the gene from which the duplication arose [13]. It is hypothesized that, over time, these fold-back structures mutate and drift away from perfect extended hairpins, with only selective pressure to retain a short fold-back structure. This fold-back structure is then acted upon by the miRNA biogenesis machinery and can efficiently regulate genes that share sequence complementarity. In 2005, it was suggested that TEs play a role in the formation of these new inverted duplications through the observation that particular inverted repeats derive from TE sequences [14]. Additionally, many TE loci simultaneously encode both siRNAs and predicted miRNAs [15], which may support the model of TE-driven miRNA evolution, because inverted repeats are initially processed into endo-siRNAs and are gradually refined over time into miRNAs. The inverted repeat model of hairpin miRNA evolution was further developed by the discovery and study of short palindromic TEs known as miniature inverted-repeat TEs (MITEs) in humans and plants [16–19]. Their short length, palindromic nature, and location in transcriptionally active regions of the genome make MITEs ideal progenitors for new miRNA genes. A recent publication demonstrated that MITE-derived endo-siRNAs regulate the abiotic
0168-9525/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2012.09.001 Trends in Genetics, December 2012, Vol. 28, No. 12
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Box 1. TE repression by small RNAs in Arabidopsis and Drosophila
Box 2. Gene-regulating small RNAs in Arabidopsis and Drosophila
The production of TE endogenous heterochromatic siRNAs and their maintenance of silenced target loci has been well characterized in the reference plant Arabidopsis (reviewed in [69]) (Table 1, main text). The process begins when the plant-specific RNA polymerase, Pol IV, transcribes through heterochromatic TE loci. This Pol IV nonprotein-coding transcript is acted upon by an RNA-dependent RNA polymerase, RDR2, producing a double-stranded RNA (dsRNA) molecule, which serves as a substrate for Arabidopsis thaliana DICER-LIKE protein 3 (AtDCL3). AtDCL3 processes the dsRNA substrate into short dsRNA molecules of the signature 24 nucleotide (nt) size, and one of these strands is incorporated into an ARGONAUTE (AGO) protein [70]. SiRNA incorporation into an AGO complex and its interaction with a Pol V scaffolding transcript results in epigenetic repression of the TE locus through the recruitment of de novo DNA methylation and histone tail-modifying proteins [71]. In Drosophila, the regulation of TEs differs depending on the cellular context. In somatic tissues, endo-siRNAs are predicted to play a role in genome defense and TE repression, because flies that lack components necessary for endo-siRNA biogenesis have increased levels of TE transcripts [72]. Endo-siRNAs are dependent on DmDicer-2, Loquacious, and DmAgo2 for their biogenesis, and DmAgo2-bound TE-derived endo-siRNAs are present in somatic and germline tissues [73–75]. In the Drosophila gonad, piRNAs are the primary small RNAs responsible for repressing TEs. The piRNA pathway produces Dicer-independent small RNAs from mostly TEderived genomic clusters and targets TEs for repression. However, a few piRNAs are not derived from TEs and target genes (reviewed in [76]). In the Drosophila ovary, the piRNA pathway is active in both the follicle cells (somatic support cells surrounding the egg chamber) and the nurse cells, which are connected to the egg cell by cytoplasmic bridges and deposit their cytoplasm, including piRNAs, into the growing egg (Figure 1a, main text). These piRNAs deposited into the egg cell are considered maternally inherited, because they originate in somatic maternal cells. PiRNA biogenesis pathways vary depending on cellular context. The somatic piRNA pathway is not amplified and utilizes only Piwi, the founding member of the PIWI clade of Argonaute proteins. By contrast, the germline pathway can amplify piRNAs through the ping-pong pathway and the utilization of two other PIWI family members, Aub and DmAgo3 [77]. These germline piRNAs play a role in silencing TEs in the next generation [49], although the exact mechanism for this silencing is not known. Current explanations include Piwi-mediated post-transcriptional cleavage of TE mRNAs, in addition to induction of heterochromatic histone modifications at TE loci (reviewed in [2]).
In contrast to TE-regulating small RNAs, gene-regulating small RNAs, such as miRNAs and the plant-specific tasiRNAs, are derived from single-copy or low-copy regions of the genome and are processed by different pathways compared with TEs (Table 1, main text). MiRNAs are transcribed by RNA polymerase (Pol) II from their own loci in Arabidopsis and Drosophila, or from genic introns in Drosophila, and then fold into hairpin precursors. In Drosophila, these structures are then processed by Drosha-Pasha to yield shorter precursor miRNAs. These are exported into the cytoplasm and cleaved by DmDicer1 into shorter dsRNA duplexes, in which one strand is eventually incorporated into DmAgo1 [3]. In Arabidopsis, AtDCL1 performs all of the pre-miRNA processing due to the lack of homologs of Drosha-Pasha, and miRNAs are loaded primarily into AtAGO1 as 21-nt RNA species (reviewed in [30]). tasiRNAs are plant-specific gene-regulating siRNAs produced from a non-protein-coding transcript. This transcript is targeted and cleaved by a miRNA, and then used as a template for an RNAdependent RNA polymerase amplification step via RDR6 and subsequent dsRNA cleavage by AtDCL4. Although they differ mechanistically in their biogenesis and initiation [78,79], 21-nt tasiRNAs and miRNAs are both incorporated into AtAGO1 and function in a similar manner to regulate genic mRNA accumulation and translational efficiency.
stress response in rice [20]. This article provided strong experimental evidence of the interaction between MITE endo-siRNAs and their putative target, because phenotypic alterations were induced by ectopically expressing these MITE siRNAs. Over time, this MITE locus may be trimmed
down to a miRNA locus, making the original MITE no longer recognizable. In addition, TEs are known to incorporate gene fragments in a process termed ‘trans-duplication’ [21], providing a mechanism for acquisition of new gene fragments from which inverted duplications can be generated. For many of the TE-derived small RNAs described in the literature [16,18,19,22], the authors make strong arguments for the ability of these molecules to regulate various candidate target genes. These arguments are based on sequence complementarity and the observation that TE small RNA accumulation often negatively correlates with the accumulation of the target mRNA. However, in each of these examples, the direct experimental interaction of these small RNAs with their predicted target mRNAs has not been conclusively demonstrated. Two new studies identify TE small RNAs that directly regulate genic mRNAs The field has recently moved beyond correlations with the publication of two new studies that experimentally define the direct regulation of a host gene mRNA by TE small RNAs. The first describes a new developmental role for maternal TE-derived piRNAs in the Drosophila embryo [23]. In the early Drosophila embryo, maternally deposited
Table 1. Distinction of small RNA types discussed in this review article Type of small RNA miRNA
Kingdom identified Animalia, Plantae
Target transcript Genic mRNAs
Endo-siRNA
Animalia, Plantae
tasiRNA
Plantae
Pseudogenes and genomic parasites, such as viruses and TEs Genic mRNAs
Heterochromatic siRNA piRNA
Plantae, Fungi Animalia
Silenced regions of the genome, such as centromeric repeats and silenced TEs TEs
Mechanism of action Post-transcriptional target degradation or translational inhibition Post-transcriptional target degradation
Refs [3]
Post-transcriptional target degradation or translational inhibition Establishment of heterochromatic marks, such as DNA and/or histone methylation Post-transcriptional target degradation and establishment of heterochromatic marks, such as DNA and/or histone methylation
[81,82]
[3,80]
[69,83] [76,84]
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through the post-transcriptional regulation of a non-TE mRNA. A second recent investigation [25] demonstrated that the epigenetic reactivation of a TE (via loss of DNA methylation and repressive histone modifications) directly regulates a genic mRNA. In nearly every tissue of the diploid phase of the wild-type Arabidopsis life cycle, TEs are efficiently epigenetically silenced (Figure 2a) and only produce 24-nt heterochromatic siRNAs (Box 1; reviewed in [1,26]). TE reactivation and RNA polymerase II (Pol II) transcription of TEs only occurs at a few distinct developmental time points in Arabidopsis (wild-type pollen, for example), as well as in plants harboring mutations in genes that play an essential role in heterochromatin condensation and epigenetic transcriptional repression [27,28]. Upon transcriptional activation and Pol II transcription, some TE transcripts, including the very abundant Athila family of long terminal repeat (LTR) retrotransposons, are degraded into 21- and 22-nt endosiRNAs [25,29]. These endo-siRNAs are processed by RNADEPENDENT RNA POLYMERASE 6 (RDR6), Arabidopsis thaliana DICER-LIKE 2 and 4 (AtDCL2/4), and A. thaliana ARGONAUTE 1 (AtAGO1), the same pathway responsible for production of trans-acting (ta)siRNAs and viral endo-siRNAs (Box 2, Table 1) [25] (reviewed in [30– 33]). At least one of these Athila endo-siRNAs (siRNA854) is incorporated into the main gene-regulating ARGONAUTE protein in Arabidopsis, AtAGO1 (Figure 2b). AtAGO1 also binds the canonical gene-regulating miRNAs and tasiRNAs to post-transcriptionally downregulate genic mRNA, a function not yet assigned to TE endosiRNAs (Box 2). Before this study, siRNA854 was only computationally predicted to target the host gene UPB1b, a plant homolog of the well-studied TIA-1 animal stress
nanos (nos) mRNA is distributed throughout the cytoplasm. Repression of nos in the prospective somatic cytoplasm of the embryo is essential for proper segmentation, because only nos mRNA at the posterior pole is translated, establishing the posterior-to-anterior Nos protein gradient necessary for proper embryonic patterning. It was previously known that an RNA-binding protein called Smaug recruits the C-C chemokine receptor type 4-negative on TATA (CCR4-NOT) deadenylase complex onto maternal nos mRNA. This recruitment is necessary for the deadenylation, translational repression, and subsequent degradation of nos mRNA in the maternal-to-zygotic transition of gene expression [24]. However, this report [23] demonstrated that the piRNA pathway also plays a role in the deadenylation and destabilization of nos mRNA (Figure 1b), based on the finding that nos mRNA is stabilized when piRNA biogenesis is perturbed. Further investigation showed that both Aubergine (Aub) and Drosophila melanogaster Ago3 (DmAgo3) proteins, which are involved in piRNA processing and activity (Box 2), are found in complex with Smaug and CCR4, and piRNAs that target the 30 UTR of the nos mRNA co-immunoprecipitate with Aub. These Aub-bound piRNAs that target the nos 30 UTR are derived from the 412 and roo TEs. A series of experiments demonstrated that both the binding sites and the accumulation of these specific piRNAs are important for nos mRNA deadenylation and repression, even though the nos mRNA shows only 17 nucleotides (nt) of complementarity to these 26-nt TE-derived piRNAs. Based on these data, the authors present a model in which maternally deposited 412 and roo-derived piRNAs aid in recruiting Smaug and the CCR4-NOT deadenylase complex to the nos mRNA via complementary binding sites in the nos 30 UTR. Thus, these TE-derived piRNAs play a role in embryonic development by contributing to the nos mRNA gradient in the early embryo
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Figure 1. Gene regulation by Drosophila transposable element (TE)-derived PIWI-interacting small RNAs (piRNAs). (a) The piRNA pathway is active in the follicle cells, nurse cells, and the egg cell of the Drosophila egg chamber. Nurse cells are connected to the growing egg cell by cytoplasmic bridges and, throughout development, the nurse cells deposit their cytoplasmic contents into the egg cell. Active TE transcripts (black) in the nurse and egg cells are processed into piRNAs (brown dashes) through the activity of Piwi, Aubergine (Aub), and Drosophila melanogaster Argonaute 3 (dmAgo3) (red). Upon fertilization, these TE-derived piRNAs are maternally inherited in the embryo. (b) In the early embryo, roo and 412 TE-derived piRNAs (brown dashes) are bound by Aub and DmAgo3 and act to repress the maternally inherited nanos mRNA through specific binding sites in its 30 untranslated region (UTR). Smaug and piRNA binding to nanos mRNA aid in the recruitment of the C-C chemokine receptor type 4-negative on TATA (CCR4-NOT) deadenylase complex and subsequent translational inhibition. Some nanos mRNA at the posterior pole of the embryo (green circle) escapes deadenylation and translational repression.
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x LTR
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RDR6, DCL4, DCL2 siRNA854 in AGO1
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Figure 2. Gene regulation by an Arabidopsis transposable element (TE)-derived small interfering (si)RNA. (a) When epigenetically silenced by DNA methylation (black circles) in a wild-type background, the Athila long terminal repeat (LTR) retrotransposon does not produce Athila-derived 21- and 22-nucleotide (nt) siRNAs. Therefore, the genic OLIGOURIDYLATE BINDING PROTEIN 1B (UBP1b) mRNA is neither affected nor regulated by Athila. (b) When epigenetic silencing is removed from Athila in wild-type pollen or a mutant context, the element is transcribed (black transcript) and, through the activity of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICER-LIKE 2 and 4 (AtDCL2/4), is processed into 21- and 22-nt endo-siRNAs. One of these Athila siRNAs, siRNA854 (brown dash), is incorporated into ARGONAUTE 1 (AtAGO1; yellow) and regulates the UBP1b mRNA through four binding sites in its 30 UTR, inhibiting UBP1b translation.
granule protein [34]. Upon further investigation [25], the post-transcriptional and translational repression of the UBP1b mRNA was found to be dependent on the accumulation of the 21- and 22-nt forms of siRNA854, as well as on the regions of the UBP1b 30 UTR that are partially complementary to siRNA854, thus demonstrating that this TE-derived endo-siRNA can directly target the UBP1b mRNA transcript (Figure 2b) [25]. The UBP1b protein is a component of plant stress granules [35], and mutants are sensitive to certain abiotic stresses [25]. Plants with epigenetically active TEs produce 21- and 22-nt siRNA854, which target the UBP1b mRNA for repression, and phenocopy the ubp1b stress-sensitive phenotype, providing a direct connection between TE epigenetic status and the host stress response. How many genes are post-transcriptionally regulated by TE small RNAs? Based on the current confirmed examples, could TE small RNAs contribute to gene regulation on a larger scale? In Drosophila, other candidate targets for regulation by abundant maternally inherited TE-derived piRNAs were predicted using an approach that combined known maternally unstable mRNAs and a relaxed criteria algorithm for piRNA targeting [23]. This study identified putative piRNA-targeted mRNAs, including hsp83 (an ATPase chaperone), oskar (a P-granule component with roles in mRNA stability), grapes (a protein kinase involved in DNA damage checkpoint control), and me31B (involved in mRNA processing bodies) (Figure 3a). The piRNA pathway was shown to play a role in the destabilization of these other Smaug-regulated maternal mRNAs in the early embryo, supporting a role for an additional level of regulation by piRNAs [23]. However, unlike UBP1b and nos, direct regulation has not been formally demonstrated. Lastly, from this analysis, as well as that of UPB1b, it is interesting to speculate that mRNA processing and
stability factors are common targets of TE small RNAs; however, the identified pool of target mRNAs needs to be expanded to determine whether this is merely a coincidence or whether there is a mechanism for the specific targeting of mRNA processing factors. It has been demonstrated that both canonical generegulating (AtAGO1) and non gene-regulating Argonaute proteins (Aub and DmAgo3) can utilize TE small RNAs for the regulation of genic mRNAs. Therefore, it is important to consider what prevents TE-derived small RNAs with the potential to be bound by Argonaute proteins from regulating hundreds or thousands of genes that share partial sequence complementarity to these small RNAs. If this were the case, upon large-scale post-transcriptional degradation of TEs, large numbers of TE piRNAs or endo-siRNAs could overwhelm gene regulatory networks. By contrast, only a subset of genes may be regulated in this manner, either due to the exclusion of TE small RNAs by AGO complexes, or a lack of opportunity for interaction with complementary targets. Recent data suggest the existence of two pools of AtAGO1 in Arabidopsis, separated by either subcellular localization or interaction with different cofactors. One AtAGO1 pool may bind miRNAs and regulate genes, whereas another pool binds siRNAs [36]. This may limit the ability for TE small RNAs to be bound by a gene-regulating pool of AGO1, ultimately limiting the number of genes post-transcriptionally regulated by TE siRNAs. However, any mechanism for fine-tuning the number and type of transcripts regulated by TE small RNAs, if it exists, is currently unknown. Regardless, there is at least the potential for a global alteration of posttranscriptional and translational gene regulation based on the high quantities of piRNAs or 21-nt endo-siRNAs from transcriptionally active TEs (Figure 3). This model may account for phenotypes in mutants with epigenetically active TEs, as well as in environmental or developmental conditions of decondensed heterochromatin. 619
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Drosophila early embryo maternal piRNAs
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hsp83 mRNA oskar mRNA
Intact nanos mRNA AAAAAA
AAAAAA
me31B mRNA
AAAAAA
AAAAAA
grapes mRNA
AAAAAA
AAAAAA
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Athila retrotransposon
LTR
RDR6, DCL4, DCL2
UBP1b mRNA
AAAAAA AAAAAA
AAAAAA AAAAAA
AAAAAA
AAAAAA
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Figure 3. Potential gene regulation by many transposable element (TE)-derived small RNAs. (a) A model for widespread gene regulation by TE-derived PIWI-interacting small RNAs (piRNAs) in Drosophila predicts that maternally inherited piRNAs (brown dashes) in the early Drosophila embryo may target other genic mRNAs. This includes both predicted (oskar, me31B, hsp83, and grapes) and unknown mRNA targets that share partial sequence complementarity. (b) A model of global gene regulation by TE-derived small interfering (si)RNAs in Arabidopsis predicts that a gene-regulating ARGONAUTE (AGO) protein, such as AtAGO1 (yellow), binds many Athila-derived siRNAs (brown dashes). This would allow these siRNAs the opportunity to regulate many other transcripts through transcript cleavage or translational inhibition via imperfect complementarity.
In addition, the piRNA pathway is essential for spermatogenesis and fertility in the mouse male germline (reviewed in [37]); therefore, it is tempting to speculate that specific piRNAs may play a role in regulating genes necessary for spermatogenesis, similar to the role seen for maternal piRNAs in the development of the Drosophila embryo. Below, we consider the possibility of this type of regulation in species with naturally active TEs. Organisms with active TEs In both Arabidopsis and Drosophila, TE activity and production into small RNAs is strictly developmentally regu620
lated [28,38–42], leading to the developmental regulation of the genes UBP1b and nos [23,25]. Other organisms, such as maize and potentially humans, have TEs that are significantly more transcriptionally active than those in laboratory strains of Arabidopsis or Drosophila [43,44], and these TEs may play a larger role in gene regulation compared with those in Arabidopsis or Drosophila. The fixation of a particular epigenetic state may cause unintended transcriptomic and phenotypic changes by affecting gene regulation through TE processing into small RNAs. For example, in maize, lines have been bred for either active or inactive TEs, fixing additional unintended traits
Review in the population [45–47]. In these maize lines, up to a quarter of the transcriptome is altered by the presence or absence of just one active TE family [48]. In various organisms, multiple scenarios induce changes in epigenetic status, such as activation of TEs by stress or hybrid formation. We conjecture that the differential production of TE small RNAs could account for dysgenesis, sterility, heterosis, or other phenotypes that emerge with changes in epigenetic status [49–53]. Additionally, TE content and activity is quickly evolving and often differs between populations of the same species [54–56], and the changes in TEderived small RNAs may contribute to some of the variation in gene regulation among taxa or between individuals. The idea that TEs are able to regulate genes from a distance is similar to Barbara McClintock’s observations of developmental consequences of TE activity [57]. McClintock referred to TEs as ‘controlling elements’ [57], and the regulation of genes by TE small RNAs provides at least one molecular mechanism by which this genic control could take place. McClintock also hypothesized that ‘genome shock’ activates TEs [58]. It is this epigenetic activation and transcription that we speculate will generate new classes of TE small RNAs that may be able to regulate partially complementary genic mRNAs. Evolution of gene-regulating TE small RNAs Although there are currently only two experimentally verified known examples of TE small RNAs regulating genic mRNAs, we imagine two distinct possibilities for the evolutionary origin of gene regulation by TE small RNAs. First, these TE small RNAs may have arisen stochastically and, therefore, the sequence complementarity between the TEderived small RNA and genic target mRNA is a coincidence. If this were the case, we predict that conserved gene regulation by TE small RNAs would be a rare event. Second, through homology or probably a duplication event, a TE may copy and incorporate a fragment of a gene through transduplication, producing TE-initiated small RNAs from a genic fragment. Either way, once a regulatory outcome exists for the interaction between TE-derived small RNAs and a genic mRNA exists, natural selection may act upon it, selecting for the sequence conservation of a short stretch of sequence similarity between the TE and the regulated gene. However, at this point, it is not possible to decipher on what level natural selection is acting, because selection may favor either a TE-driven process that results in a replicative advantage for the TE, or a host-driven process resulting in co-option or suppression of the TE. Below, we consider and offer our ideas on the possibility of both TE-driven and hostdriven selection. TE-driven evolution The targeting of genes by TE-derived small RNAs may be a strategy that provides the TE with a survival advantage. TEs and their host genomes are often thought to be engaged in an evolutionary ‘arms-race’, with the TE attempting to gain activity while the host genome attempts to suppress this activity. As the arms-race escalates, each side creates or steals new mechanisms to aid their struggle. Therefore, we speculate that a TE strategy for retaining activity may involve the acquisition of genic sequences as
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‘hostages’ [59]. If the TE acquires a piece of the genic sequence and is then processed into small RNAs, some of these small RNAs bear complementarity to the host gene. Therefore, the TE makes the host pay a penalty for silencing the TE, because silencing of the TE transcripts via degradation into small RNAs will result in silencing of the host endogenous gene in trans. It would be particularly useful for the TE to capture and regulate host genes that are directly responsible for silencing the TE, effectively neutralizing the ability of the host to use these weapons to repress the TE. In this way, TE small RNAs may serve as silencing suppressors, actively targeting genes that repress them. There is some indirect evidence to suggest that TEs use small RNAs to regulate host genes that are repressing TE activity. For example, other post-transcriptionally regulated targets of TE-derived piRNAs have been predicted in the early Drosophila embryo [23]. One of these predicted targets, Me31B, is a P-body component of the nuage, a germlinespecific dense perinuclear structure. A recent report demonstrated that Me31B P-body complexes are involved in the repression of TE activity in the nuage [60]. Based on this role of Me31B, TE piRNAs that target this mRNA may do so to suppress host silencing. Additionally, the Arabidopsis TEderived siRNA854 may regulate UBP1b mRNA to suppress host silencing and provide an advantage to the TE. Recent data suggest that the UBP1b protein senses the intracellular stress caused by loss of heterochromatin and active TEs [25]. In light of its role as a component of stress granules [35] (ribonucleoprotein granules that sequester a subpopulation of the cytoplasmic mRNA away from polysomes), it is tempting to speculate that UBP1b may play a role in the sequestration of TE transcripts, preventing the translation of their mRNA into proteins necessary for TE transposition. This is similar to the role of the mammalian homolog of UBP1b, TIA-1 [61], because TIA-1 can repress the activity of particular viruses and retrotransposons through the formation of stress granules (reviewed in [62]). Therefore, the evolution of these TE-derived small RNAs would serve an advantage to the TE and would explain why these TEs have retained specific small RNA sequences. By contrast, in the absence of experimentation and data, there is an equally valid argument that UBP1b could promote TE activity, because there are known examples in the literature of stress granule formation and the TIA-1 protein aiding in viral replication [63–65]. If this is the case, the retention of TE small RNA sequences that target UBP1b for downregulation may be a host-driven process used in the ongoing arms-race for the repression of TEs to maintain genomic integrity. Experimental demonstration of TE repression by these TE-regulated host genes is needed to demonstrate this theory directly. Host-driven evolution It is possible that the processing of TE transcripts into small RNAs that regulate genes may confer a selective advantage to the host in the arms-race between host and TE. We speculate that gene regulation by TE-derived small RNAs may represent an example of the host co-opting the TE for its own purposes. Although TEs evolved to replicate at the expense of the host genome, certain TEs may be 621
Review retained for the regulation of genes under particular conditions, using the epigenetic regulation of TEs to ‘store’ small RNA information. In this way, small RNAs accumulate at the correct developmental time, place, or stress condition where this gene regulatory information is needed. For example, in the early Drosophila embryo, the targeting of maternal nos mRNA for degradation by TE-derived piRNAs appears to be an instance in which a burst of TE activity at a particular developmental time point aids the host in fine-tuning an important cellular function. The importance of the interaction between 412 and roo-derived piRNAs and their nos mRNA binding sites is clear, because abolishing this interaction with anti-piRNAs results in head developmental defects [23]. Circumstantial evidence of a host-driven process in Arabidopsis in which TEs store small RNA information comes from the overlap between genome-wide transcriptional changes upon stress and known TE activation upon the same stress conditions [66,67]. However, it is possible that TE small RNAs are merely a byproduct of transient TE expression and do not contribute to genome-wide transcriptional changes. Experiments that demonstrate the reduced ability of an organism to overcome stress due to a lack of TE small RNAs are required to assess this theory. Concluding remarks Although bioinformatic approaches have predicted that relationships between TE-derived small RNAs and genic mRNAs are prevalent, to date only two recent publications experimentally verify this mode of regulation. There are many questions that remain, even in these two characterized examples. For instance, the exact mechanism of regulation by TE small RNAs is still unclear, because although one study [23] demonstrated the necessity of the piRNA binding sites in the nos 30 UTR for nos repression, others have shown that these binding sites are not required for rescue of the nos phenotype [68]. It is possible that the TE-derived piRNAs play more of a fine-tuning role in nos mRNA regulation than a Smaug-recruitment role. Additionally, another study [25] reported that the TE-derived siRNA854 could regulate UBP1b mRNA through translational inhibition in one tissue and through transcript cleavage in another. Again, the exact mechanism of regulation in this instance is not clear. It is impossible to determine the scope, evolution, and role of this generegulatory mechanism based on two examples. However, it is now apparent that the categorical division between the regulation of genes and the production of TE small RNAs is not as distinct in vivo, and it is likely that characterization of additional examples will follow. These future examples should further reveal the oftenoverlooked influences of TE activity on the genic transcriptome. Acknowledgments The authors thank Robin Wharton for critically reading this manuscript. A.D.M. is supported by a fellowship from The Ohio State University Center for RNA Biology. Research in the Slotkin laboratory is supported by National Science Foundation (NSF) grant MCB-1020499.
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58 McClintock, B. (1984) The significance of responses of the genome to challenge. Science 226, 792–801 59 Lisch, D. and Slotkin, R.K. (2011) Strategies for silencing and escape: the ancient struggle between transposable elements and their hosts. Int. Rev. Cell Mol. Biol. 292, 119–152 60 Liu, L. et al. (2011) PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition. Development 138, 1863–1873 61 Kedersha, N.L. et al. (1999) RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF–2 alpha to the assembly of mammalian stress granules. J. Cell Biol. 147, 1431–1442 62 Beckham, C.J. and Parker, R. (2008) P bodies, stress granules, and viral life cycles. Cell Host Microbe 3, 206–212 63 Li, W. et al. (2002) Cell proteins TIA-1 and TIAR interact with the 30 stem-loop of the West Nile Virus complementary minus-strand RNA and facilitate virus replication. J. Virol. 76, 11989–12000 64 Qin, Q. et al. (2009) Mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection. J. Virol. 83, 11090–11101 65 Lindquist, M.E. et al. (2010) Respiratory syncytial virus induces host RNA stress granules to facilitate viral replication. J. Virol. 84, 12274– 12284 66 Zeller, G. et al. (2009) Stress-induced changes in the Arabidopsis thaliana transcriptome analyzed using whole-genome tiling arrays. Plant J. 58, 1068–1082 67 Ito, H. et al. (2011) An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472, 115–119 68 Dahanukar, A. and Wharton, R.P. (1996) The Nanos gradient in Drosophila embryos is generated by translational regulation. Genes Dev. 10, 2610–2620 69 Law, J.A. and Jacobsen, S.E. (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 1–17 70 Qi, Y. et al. (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012 71 Wierzbicki, A.T. et al. (2008) Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135, 1–14 72 Chung, W. et al. (2008) Endogenous RNA interference provides a somatic defense against Drosophila transposons. Curr. Biol. 18, 795–802 73 Ghildiyal, M. et al. (2008) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320, 1077–1081 74 Czech, B. et al. (2008) An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798–802 75 Kawamura, Y. et al. (2008) Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793–797 76 Siomi, M.C. et al. (2011) PIWI-interacting small RNAs: the vanguard of genome defence. Nat. Rev. Mol. Cell Biol. 12, 246–258 77 Senti, K. et al. (2010) The piRNA pathway: a fly’s perspective on the guardian of the genome. Trends Genet. 26, 499–509 78 Vazquez, F. et al. (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79 79 Allen, E. et al. (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 80 Ding, S. (2010) RNA-based antiviral immunity. Nat. Rev. Immunol. 10, 632–644 81 Felippes, F.F. and Weigel, D. (2009) Triggering the formation of tasiRNAs in Arabidopsis thaliana: the role of microRNA miR173. EMBO Rep. 10, 1–7 82 Montgomery, T.A. et al. (2008) Specificity of ARGONAUTE7-mir390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 83 Chang, S. et al. (2012) RNA interference pathways in fungi: mechanisms and functions. Annu. Rev. Microbiol. 66, 305–323 84 Senti, K. and Brennecke, J. (2010) The piRNA pathway: a fly’s perspective on the guardian of the genome. Trends Genet. 26, 499–509
Further reading Malone, C.D. and Hannon, G.J. (2009) Small RNAs as guardians of the genome. Cell 136, 656–668 623
Transposable element small RNAs as regulators of gene expression Andrea D. McCue and R. Keith Slotkin Department of Molecular Genetics & Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Transposable elements (TEs) are a source of endogenous small RNAs in animals and plants. These TE-derived small RNAs have been traditionally treated as functionally distinct from gene-regulating small RNAs, such as miRNAs. Two recent reports in Drosophila and Arabidopsis have blurred the lines of this distinction. In both examples, epigenetically and developmentally regulated bursts in TE expression produce gene-regulating small RNAs. In the Drosophila early embryo, maternally deposited TEderived PIWI-interacting small RNAs (piRNAs) play a role in regulating the nanos mRNA through small RNA binding sites in the nanos 30 untranslated region (UTR). In Arabidopsis, when Athila retrotransposons are epigenetically activated, their transcripts are processed into small RNAs, which directly target the 30 UTR of the genic oligouridylate binding protein 1B (UBP1b) mRNA. Based on these two examples, we suggest that other TE-derived small RNAs regulate additional genes and propose that, through small RNAs, the epigenetic status of TEs could widely influence the genic transcriptome. The long-held distinction between gene-regulating and TE-regulating small RNAs The regulation of genes has long been treated as an independent pathway from the regulation of TEs. Even as new examples of genes or genome-wide processes that are regulated by TEs appear in publication almost monthly, current dogma still separates the regulation of the relatively clean single-copy genes from the messy repetitive genome context in which they reside. One such long-standing functional separation between TE and gene has been in the classes of small RNAs they produce. The literature describing small RNA biology has generally categorized distinctly separate small RNA biogenesis pathways for the regulation of repetitive, TE-rich regions of the genome (Box 1) and for the regulation of genes (Box 2) [1–3]. TEs are major producers of endogenous small interfering RNAs (endo-siRNAs) in plants and animals and piRNAs in animals (Table 1). These two classes of small RNAs act to repress TE mRNA accumulation post-transcriptionally (reviewed in [2]) and, in some cases, to induce DNA methylation and repressive histone tail modifications at TE loci to maintain the element in a transcriptionally repressed heterochromatic state [4–7]. TE regulation has long been known to influence proximal neighboring genes by readthrough transcription or ectopic recruitment of silencing Corresponding author: Slotkin, R.K. ([email protected]) Keywords: transposable element; small RNAs; piRNA; siRNA; gene regulation.
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factors (reviewed in [1,8]). However, two recently published examples use fundamentally distinct mechanisms whereby host genes regulated by the TE do not have to be adjacent to or associated with any TE fragment. In these cases, a TE-derived small RNA acts in trans in a similar fashion as a miRNA, post-transcriptionally or translationally repressing target genes with imperfect complementarity. These two cases blur the lines between the wellengrained distinction of gene-regulating and TE-derived small RNAs. Initial inquiries into gene-regulating TE small RNAs The idea that TE-derived small RNAs could regulate genes is not new. Several virus-derived miRNAs were identified in 2004 [9], and it is likely that these miRNAs post-transcriptionally alter host gene expression [10,11]. In 2005, miRNAs were also predicted from mammalian genomic repeat sequences [12], suggesting that, similar to viruses, TE miRNAs could influence genes in a post-transcriptional manner. TEs may also play a role in the evolution of miRNAs. miRNA evolution is postulated to initiate from short inverted duplications of genes, because these inverted duplications could form extended hairpin foldback structures that are acted upon by Dicer proteins, producing endo-siRNAs that have the ability to target the gene from which the duplication arose [13]. It is hypothesized that, over time, these fold-back structures mutate and drift away from perfect extended hairpins, with only selective pressure to retain a short fold-back structure. This fold-back structure is then acted upon by the miRNA biogenesis machinery and can efficiently regulate genes that share sequence complementarity. In 2005, it was suggested that TEs play a role in the formation of these new inverted duplications through the observation that particular inverted repeats derive from TE sequences [14]. Additionally, many TE loci simultaneously encode both siRNAs and predicted miRNAs [15], which may support the model of TE-driven miRNA evolution, because inverted repeats are initially processed into endo-siRNAs and are gradually refined over time into miRNAs. The inverted repeat model of hairpin miRNA evolution was further developed by the discovery and study of short palindromic TEs known as miniature inverted-repeat TEs (MITEs) in humans and plants [16–19]. Their short length, palindromic nature, and location in transcriptionally active regions of the genome make MITEs ideal progenitors for new miRNA genes. A recent publication demonstrated that MITE-derived endo-siRNAs regulate the abiotic
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Box 1. TE repression by small RNAs in Arabidopsis and Drosophila
Box 2. Gene-regulating small RNAs in Arabidopsis and Drosophila
The production of TE endogenous heterochromatic siRNAs and their maintenance of silenced target loci has been well characterized in the reference plant Arabidopsis (reviewed in [69]) (Table 1, main text). The process begins when the plant-specific RNA polymerase, Pol IV, transcribes through heterochromatic TE loci. This Pol IV nonprotein-coding transcript is acted upon by an RNA-dependent RNA polymerase, RDR2, producing a double-stranded RNA (dsRNA) molecule, which serves as a substrate for Arabidopsis thaliana DICER-LIKE protein 3 (AtDCL3). AtDCL3 processes the dsRNA substrate into short dsRNA molecules of the signature 24 nucleotide (nt) size, and one of these strands is incorporated into an ARGONAUTE (AGO) protein [70]. SiRNA incorporation into an AGO complex and its interaction with a Pol V scaffolding transcript results in epigenetic repression of the TE locus through the recruitment of de novo DNA methylation and histone tail-modifying proteins [71]. In Drosophila, the regulation of TEs differs depending on the cellular context. In somatic tissues, endo-siRNAs are predicted to play a role in genome defense and TE repression, because flies that lack components necessary for endo-siRNA biogenesis have increased levels of TE transcripts [72]. Endo-siRNAs are dependent on DmDicer-2, Loquacious, and DmAgo2 for their biogenesis, and DmAgo2-bound TE-derived endo-siRNAs are present in somatic and germline tissues [73–75]. In the Drosophila gonad, piRNAs are the primary small RNAs responsible for repressing TEs. The piRNA pathway produces Dicer-independent small RNAs from mostly TEderived genomic clusters and targets TEs for repression. However, a few piRNAs are not derived from TEs and target genes (reviewed in [76]). In the Drosophila ovary, the piRNA pathway is active in both the follicle cells (somatic support cells surrounding the egg chamber) and the nurse cells, which are connected to the egg cell by cytoplasmic bridges and deposit their cytoplasm, including piRNAs, into the growing egg (Figure 1a, main text). These piRNAs deposited into the egg cell are considered maternally inherited, because they originate in somatic maternal cells. PiRNA biogenesis pathways vary depending on cellular context. The somatic piRNA pathway is not amplified and utilizes only Piwi, the founding member of the PIWI clade of Argonaute proteins. By contrast, the germline pathway can amplify piRNAs through the ping-pong pathway and the utilization of two other PIWI family members, Aub and DmAgo3 [77]. These germline piRNAs play a role in silencing TEs in the next generation [49], although the exact mechanism for this silencing is not known. Current explanations include Piwi-mediated post-transcriptional cleavage of TE mRNAs, in addition to induction of heterochromatic histone modifications at TE loci (reviewed in [2]).
In contrast to TE-regulating small RNAs, gene-regulating small RNAs, such as miRNAs and the plant-specific tasiRNAs, are derived from single-copy or low-copy regions of the genome and are processed by different pathways compared with TEs (Table 1, main text). MiRNAs are transcribed by RNA polymerase (Pol) II from their own loci in Arabidopsis and Drosophila, or from genic introns in Drosophila, and then fold into hairpin precursors. In Drosophila, these structures are then processed by Drosha-Pasha to yield shorter precursor miRNAs. These are exported into the cytoplasm and cleaved by DmDicer1 into shorter dsRNA duplexes, in which one strand is eventually incorporated into DmAgo1 [3]. In Arabidopsis, AtDCL1 performs all of the pre-miRNA processing due to the lack of homologs of Drosha-Pasha, and miRNAs are loaded primarily into AtAGO1 as 21-nt RNA species (reviewed in [30]). tasiRNAs are plant-specific gene-regulating siRNAs produced from a non-protein-coding transcript. This transcript is targeted and cleaved by a miRNA, and then used as a template for an RNAdependent RNA polymerase amplification step via RDR6 and subsequent dsRNA cleavage by AtDCL4. Although they differ mechanistically in their biogenesis and initiation [78,79], 21-nt tasiRNAs and miRNAs are both incorporated into AtAGO1 and function in a similar manner to regulate genic mRNA accumulation and translational efficiency.
stress response in rice [20]. This article provided strong experimental evidence of the interaction between MITE endo-siRNAs and their putative target, because phenotypic alterations were induced by ectopically expressing these MITE siRNAs. Over time, this MITE locus may be trimmed
down to a miRNA locus, making the original MITE no longer recognizable. In addition, TEs are known to incorporate gene fragments in a process termed ‘trans-duplication’ [21], providing a mechanism for acquisition of new gene fragments from which inverted duplications can be generated. For many of the TE-derived small RNAs described in the literature [16,18,19,22], the authors make strong arguments for the ability of these molecules to regulate various candidate target genes. These arguments are based on sequence complementarity and the observation that TE small RNA accumulation often negatively correlates with the accumulation of the target mRNA. However, in each of these examples, the direct experimental interaction of these small RNAs with their predicted target mRNAs has not been conclusively demonstrated. Two new studies identify TE small RNAs that directly regulate genic mRNAs The field has recently moved beyond correlations with the publication of two new studies that experimentally define the direct regulation of a host gene mRNA by TE small RNAs. The first describes a new developmental role for maternal TE-derived piRNAs in the Drosophila embryo [23]. In the early Drosophila embryo, maternally deposited
Table 1. Distinction of small RNA types discussed in this review article Type of small RNA miRNA
Kingdom identified Animalia, Plantae
Target transcript Genic mRNAs
Endo-siRNA
Animalia, Plantae
tasiRNA
Plantae
Pseudogenes and genomic parasites, such as viruses and TEs Genic mRNAs
Heterochromatic siRNA piRNA
Plantae, Fungi Animalia
Silenced regions of the genome, such as centromeric repeats and silenced TEs TEs
Mechanism of action Post-transcriptional target degradation or translational inhibition Post-transcriptional target degradation
Refs [3]
Post-transcriptional target degradation or translational inhibition Establishment of heterochromatic marks, such as DNA and/or histone methylation Post-transcriptional target degradation and establishment of heterochromatic marks, such as DNA and/or histone methylation
[81,82]
[3,80]
[69,83] [76,84]
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through the post-transcriptional regulation of a non-TE mRNA. A second recent investigation [25] demonstrated that the epigenetic reactivation of a TE (via loss of DNA methylation and repressive histone modifications) directly regulates a genic mRNA. In nearly every tissue of the diploid phase of the wild-type Arabidopsis life cycle, TEs are efficiently epigenetically silenced (Figure 2a) and only produce 24-nt heterochromatic siRNAs (Box 1; reviewed in [1,26]). TE reactivation and RNA polymerase II (Pol II) transcription of TEs only occurs at a few distinct developmental time points in Arabidopsis (wild-type pollen, for example), as well as in plants harboring mutations in genes that play an essential role in heterochromatin condensation and epigenetic transcriptional repression [27,28]. Upon transcriptional activation and Pol II transcription, some TE transcripts, including the very abundant Athila family of long terminal repeat (LTR) retrotransposons, are degraded into 21- and 22-nt endosiRNAs [25,29]. These endo-siRNAs are processed by RNADEPENDENT RNA POLYMERASE 6 (RDR6), Arabidopsis thaliana DICER-LIKE 2 and 4 (AtDCL2/4), and A. thaliana ARGONAUTE 1 (AtAGO1), the same pathway responsible for production of trans-acting (ta)siRNAs and viral endo-siRNAs (Box 2, Table 1) [25] (reviewed in [30– 33]). At least one of these Athila endo-siRNAs (siRNA854) is incorporated into the main gene-regulating ARGONAUTE protein in Arabidopsis, AtAGO1 (Figure 2b). AtAGO1 also binds the canonical gene-regulating miRNAs and tasiRNAs to post-transcriptionally downregulate genic mRNA, a function not yet assigned to TE endosiRNAs (Box 2). Before this study, siRNA854 was only computationally predicted to target the host gene UPB1b, a plant homolog of the well-studied TIA-1 animal stress
nanos (nos) mRNA is distributed throughout the cytoplasm. Repression of nos in the prospective somatic cytoplasm of the embryo is essential for proper segmentation, because only nos mRNA at the posterior pole is translated, establishing the posterior-to-anterior Nos protein gradient necessary for proper embryonic patterning. It was previously known that an RNA-binding protein called Smaug recruits the C-C chemokine receptor type 4-negative on TATA (CCR4-NOT) deadenylase complex onto maternal nos mRNA. This recruitment is necessary for the deadenylation, translational repression, and subsequent degradation of nos mRNA in the maternal-to-zygotic transition of gene expression [24]. However, this report [23] demonstrated that the piRNA pathway also plays a role in the deadenylation and destabilization of nos mRNA (Figure 1b), based on the finding that nos mRNA is stabilized when piRNA biogenesis is perturbed. Further investigation showed that both Aubergine (Aub) and Drosophila melanogaster Ago3 (DmAgo3) proteins, which are involved in piRNA processing and activity (Box 2), are found in complex with Smaug and CCR4, and piRNAs that target the 30 UTR of the nos mRNA co-immunoprecipitate with Aub. These Aub-bound piRNAs that target the nos 30 UTR are derived from the 412 and roo TEs. A series of experiments demonstrated that both the binding sites and the accumulation of these specific piRNAs are important for nos mRNA deadenylation and repression, even though the nos mRNA shows only 17 nucleotides (nt) of complementarity to these 26-nt TE-derived piRNAs. Based on these data, the authors present a model in which maternally deposited 412 and roo-derived piRNAs aid in recruiting Smaug and the CCR4-NOT deadenylase complex to the nos mRNA via complementary binding sites in the nos 30 UTR. Thus, these TE-derived piRNAs play a role in embryonic development by contributing to the nos mRNA gradient in the early embryo
(a)
(b)
Egg chamber
Nurse cell nscript s TE Tra
Gr
piRNAs
ell N u rs e c e
Ferlizaon and inheritance of maternal piRNAs into the embryo
ing egg c ow
Piwi/Aub/Ago3
Early embryo
roo and 412 TE-derived piRNAs
nanos mRNA
AAAAAA
Intact nanos mRNA
Smaug Aub/Ago3 CCR4-NOT deadenylase
ll
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Figure 1. Gene regulation by Drosophila transposable element (TE)-derived PIWI-interacting small RNAs (piRNAs). (a) The piRNA pathway is active in the follicle cells, nurse cells, and the egg cell of the Drosophila egg chamber. Nurse cells are connected to the growing egg cell by cytoplasmic bridges and, throughout development, the nurse cells deposit their cytoplasmic contents into the egg cell. Active TE transcripts (black) in the nurse and egg cells are processed into piRNAs (brown dashes) through the activity of Piwi, Aubergine (Aub), and Drosophila melanogaster Argonaute 3 (dmAgo3) (red). Upon fertilization, these TE-derived piRNAs are maternally inherited in the embryo. (b) In the early embryo, roo and 412 TE-derived piRNAs (brown dashes) are bound by Aub and DmAgo3 and act to repress the maternally inherited nanos mRNA through specific binding sites in its 30 untranslated region (UTR). Smaug and piRNA binding to nanos mRNA aid in the recruitment of the C-C chemokine receptor type 4-negative on TATA (CCR4-NOT) deadenylase complex and subsequent translational inhibition. Some nanos mRNA at the posterior pole of the embryo (green circle) escapes deadenylation and translational repression.
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(a)
(b)
x LTR
Epigenecally silenced
Transcriponally acve
Athila retrotransposon
LTR
LTR
Athila retrotransposon
LTR
RDR6, DCL4, DCL2 siRNA854 in AGO1
UBP1b mRNA
AAAAAA
Translaon
UBP1b mRNA
AAAAAA
x
Translaon
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Figure 2. Gene regulation by an Arabidopsis transposable element (TE)-derived small interfering (si)RNA. (a) When epigenetically silenced by DNA methylation (black circles) in a wild-type background, the Athila long terminal repeat (LTR) retrotransposon does not produce Athila-derived 21- and 22-nucleotide (nt) siRNAs. Therefore, the genic OLIGOURIDYLATE BINDING PROTEIN 1B (UBP1b) mRNA is neither affected nor regulated by Athila. (b) When epigenetic silencing is removed from Athila in wild-type pollen or a mutant context, the element is transcribed (black transcript) and, through the activity of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICER-LIKE 2 and 4 (AtDCL2/4), is processed into 21- and 22-nt endo-siRNAs. One of these Athila siRNAs, siRNA854 (brown dash), is incorporated into ARGONAUTE 1 (AtAGO1; yellow) and regulates the UBP1b mRNA through four binding sites in its 30 UTR, inhibiting UBP1b translation.
granule protein [34]. Upon further investigation [25], the post-transcriptional and translational repression of the UBP1b mRNA was found to be dependent on the accumulation of the 21- and 22-nt forms of siRNA854, as well as on the regions of the UBP1b 30 UTR that are partially complementary to siRNA854, thus demonstrating that this TE-derived endo-siRNA can directly target the UBP1b mRNA transcript (Figure 2b) [25]. The UBP1b protein is a component of plant stress granules [35], and mutants are sensitive to certain abiotic stresses [25]. Plants with epigenetically active TEs produce 21- and 22-nt siRNA854, which target the UBP1b mRNA for repression, and phenocopy the ubp1b stress-sensitive phenotype, providing a direct connection between TE epigenetic status and the host stress response. How many genes are post-transcriptionally regulated by TE small RNAs? Based on the current confirmed examples, could TE small RNAs contribute to gene regulation on a larger scale? In Drosophila, other candidate targets for regulation by abundant maternally inherited TE-derived piRNAs were predicted using an approach that combined known maternally unstable mRNAs and a relaxed criteria algorithm for piRNA targeting [23]. This study identified putative piRNA-targeted mRNAs, including hsp83 (an ATPase chaperone), oskar (a P-granule component with roles in mRNA stability), grapes (a protein kinase involved in DNA damage checkpoint control), and me31B (involved in mRNA processing bodies) (Figure 3a). The piRNA pathway was shown to play a role in the destabilization of these other Smaug-regulated maternal mRNAs in the early embryo, supporting a role for an additional level of regulation by piRNAs [23]. However, unlike UBP1b and nos, direct regulation has not been formally demonstrated. Lastly, from this analysis, as well as that of UPB1b, it is interesting to speculate that mRNA processing and
stability factors are common targets of TE small RNAs; however, the identified pool of target mRNAs needs to be expanded to determine whether this is merely a coincidence or whether there is a mechanism for the specific targeting of mRNA processing factors. It has been demonstrated that both canonical generegulating (AtAGO1) and non gene-regulating Argonaute proteins (Aub and DmAgo3) can utilize TE small RNAs for the regulation of genic mRNAs. Therefore, it is important to consider what prevents TE-derived small RNAs with the potential to be bound by Argonaute proteins from regulating hundreds or thousands of genes that share partial sequence complementarity to these small RNAs. If this were the case, upon large-scale post-transcriptional degradation of TEs, large numbers of TE piRNAs or endo-siRNAs could overwhelm gene regulatory networks. By contrast, only a subset of genes may be regulated in this manner, either due to the exclusion of TE small RNAs by AGO complexes, or a lack of opportunity for interaction with complementary targets. Recent data suggest the existence of two pools of AtAGO1 in Arabidopsis, separated by either subcellular localization or interaction with different cofactors. One AtAGO1 pool may bind miRNAs and regulate genes, whereas another pool binds siRNAs [36]. This may limit the ability for TE small RNAs to be bound by a gene-regulating pool of AGO1, ultimately limiting the number of genes post-transcriptionally regulated by TE siRNAs. However, any mechanism for fine-tuning the number and type of transcripts regulated by TE small RNAs, if it exists, is currently unknown. Regardless, there is at least the potential for a global alteration of posttranscriptional and translational gene regulation based on the high quantities of piRNAs or 21-nt endo-siRNAs from transcriptionally active TEs (Figure 3). This model may account for phenotypes in mutants with epigenetically active TEs, as well as in environmental or developmental conditions of decondensed heterochromatin. 619
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(a)
Drosophila early embryo maternal piRNAs
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Figure 3. Potential gene regulation by many transposable element (TE)-derived small RNAs. (a) A model for widespread gene regulation by TE-derived PIWI-interacting small RNAs (piRNAs) in Drosophila predicts that maternally inherited piRNAs (brown dashes) in the early Drosophila embryo may target other genic mRNAs. This includes both predicted (oskar, me31B, hsp83, and grapes) and unknown mRNA targets that share partial sequence complementarity. (b) A model of global gene regulation by TE-derived small interfering (si)RNAs in Arabidopsis predicts that a gene-regulating ARGONAUTE (AGO) protein, such as AtAGO1 (yellow), binds many Athila-derived siRNAs (brown dashes). This would allow these siRNAs the opportunity to regulate many other transcripts through transcript cleavage or translational inhibition via imperfect complementarity.
In addition, the piRNA pathway is essential for spermatogenesis and fertility in the mouse male germline (reviewed in [37]); therefore, it is tempting to speculate that specific piRNAs may play a role in regulating genes necessary for spermatogenesis, similar to the role seen for maternal piRNAs in the development of the Drosophila embryo. Below, we consider the possibility of this type of regulation in species with naturally active TEs. Organisms with active TEs In both Arabidopsis and Drosophila, TE activity and production into small RNAs is strictly developmentally regu620
lated [28,38–42], leading to the developmental regulation of the genes UBP1b and nos [23,25]. Other organisms, such as maize and potentially humans, have TEs that are significantly more transcriptionally active than those in laboratory strains of Arabidopsis or Drosophila [43,44], and these TEs may play a larger role in gene regulation compared with those in Arabidopsis or Drosophila. The fixation of a particular epigenetic state may cause unintended transcriptomic and phenotypic changes by affecting gene regulation through TE processing into small RNAs. For example, in maize, lines have been bred for either active or inactive TEs, fixing additional unintended traits
Review in the population [45–47]. In these maize lines, up to a quarter of the transcriptome is altered by the presence or absence of just one active TE family [48]. In various organisms, multiple scenarios induce changes in epigenetic status, such as activation of TEs by stress or hybrid formation. We conjecture that the differential production of TE small RNAs could account for dysgenesis, sterility, heterosis, or other phenotypes that emerge with changes in epigenetic status [49–53]. Additionally, TE content and activity is quickly evolving and often differs between populations of the same species [54–56], and the changes in TEderived small RNAs may contribute to some of the variation in gene regulation among taxa or between individuals. The idea that TEs are able to regulate genes from a distance is similar to Barbara McClintock’s observations of developmental consequences of TE activity [57]. McClintock referred to TEs as ‘controlling elements’ [57], and the regulation of genes by TE small RNAs provides at least one molecular mechanism by which this genic control could take place. McClintock also hypothesized that ‘genome shock’ activates TEs [58]. It is this epigenetic activation and transcription that we speculate will generate new classes of TE small RNAs that may be able to regulate partially complementary genic mRNAs. Evolution of gene-regulating TE small RNAs Although there are currently only two experimentally verified known examples of TE small RNAs regulating genic mRNAs, we imagine two distinct possibilities for the evolutionary origin of gene regulation by TE small RNAs. First, these TE small RNAs may have arisen stochastically and, therefore, the sequence complementarity between the TEderived small RNA and genic target mRNA is a coincidence. If this were the case, we predict that conserved gene regulation by TE small RNAs would be a rare event. Second, through homology or probably a duplication event, a TE may copy and incorporate a fragment of a gene through transduplication, producing TE-initiated small RNAs from a genic fragment. Either way, once a regulatory outcome exists for the interaction between TE-derived small RNAs and a genic mRNA exists, natural selection may act upon it, selecting for the sequence conservation of a short stretch of sequence similarity between the TE and the regulated gene. However, at this point, it is not possible to decipher on what level natural selection is acting, because selection may favor either a TE-driven process that results in a replicative advantage for the TE, or a host-driven process resulting in co-option or suppression of the TE. Below, we consider and offer our ideas on the possibility of both TE-driven and hostdriven selection. TE-driven evolution The targeting of genes by TE-derived small RNAs may be a strategy that provides the TE with a survival advantage. TEs and their host genomes are often thought to be engaged in an evolutionary ‘arms-race’, with the TE attempting to gain activity while the host genome attempts to suppress this activity. As the arms-race escalates, each side creates or steals new mechanisms to aid their struggle. Therefore, we speculate that a TE strategy for retaining activity may involve the acquisition of genic sequences as
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‘hostages’ [59]. If the TE acquires a piece of the genic sequence and is then processed into small RNAs, some of these small RNAs bear complementarity to the host gene. Therefore, the TE makes the host pay a penalty for silencing the TE, because silencing of the TE transcripts via degradation into small RNAs will result in silencing of the host endogenous gene in trans. It would be particularly useful for the TE to capture and regulate host genes that are directly responsible for silencing the TE, effectively neutralizing the ability of the host to use these weapons to repress the TE. In this way, TE small RNAs may serve as silencing suppressors, actively targeting genes that repress them. There is some indirect evidence to suggest that TEs use small RNAs to regulate host genes that are repressing TE activity. For example, other post-transcriptionally regulated targets of TE-derived piRNAs have been predicted in the early Drosophila embryo [23]. One of these predicted targets, Me31B, is a P-body component of the nuage, a germlinespecific dense perinuclear structure. A recent report demonstrated that Me31B P-body complexes are involved in the repression of TE activity in the nuage [60]. Based on this role of Me31B, TE piRNAs that target this mRNA may do so to suppress host silencing. Additionally, the Arabidopsis TEderived siRNA854 may regulate UBP1b mRNA to suppress host silencing and provide an advantage to the TE. Recent data suggest that the UBP1b protein senses the intracellular stress caused by loss of heterochromatin and active TEs [25]. In light of its role as a component of stress granules [35] (ribonucleoprotein granules that sequester a subpopulation of the cytoplasmic mRNA away from polysomes), it is tempting to speculate that UBP1b may play a role in the sequestration of TE transcripts, preventing the translation of their mRNA into proteins necessary for TE transposition. This is similar to the role of the mammalian homolog of UBP1b, TIA-1 [61], because TIA-1 can repress the activity of particular viruses and retrotransposons through the formation of stress granules (reviewed in [62]). Therefore, the evolution of these TE-derived small RNAs would serve an advantage to the TE and would explain why these TEs have retained specific small RNA sequences. By contrast, in the absence of experimentation and data, there is an equally valid argument that UBP1b could promote TE activity, because there are known examples in the literature of stress granule formation and the TIA-1 protein aiding in viral replication [63–65]. If this is the case, the retention of TE small RNA sequences that target UBP1b for downregulation may be a host-driven process used in the ongoing arms-race for the repression of TEs to maintain genomic integrity. Experimental demonstration of TE repression by these TE-regulated host genes is needed to demonstrate this theory directly. Host-driven evolution It is possible that the processing of TE transcripts into small RNAs that regulate genes may confer a selective advantage to the host in the arms-race between host and TE. We speculate that gene regulation by TE-derived small RNAs may represent an example of the host co-opting the TE for its own purposes. Although TEs evolved to replicate at the expense of the host genome, certain TEs may be 621
Review retained for the regulation of genes under particular conditions, using the epigenetic regulation of TEs to ‘store’ small RNA information. In this way, small RNAs accumulate at the correct developmental time, place, or stress condition where this gene regulatory information is needed. For example, in the early Drosophila embryo, the targeting of maternal nos mRNA for degradation by TE-derived piRNAs appears to be an instance in which a burst of TE activity at a particular developmental time point aids the host in fine-tuning an important cellular function. The importance of the interaction between 412 and roo-derived piRNAs and their nos mRNA binding sites is clear, because abolishing this interaction with anti-piRNAs results in head developmental defects [23]. Circumstantial evidence of a host-driven process in Arabidopsis in which TEs store small RNA information comes from the overlap between genome-wide transcriptional changes upon stress and known TE activation upon the same stress conditions [66,67]. However, it is possible that TE small RNAs are merely a byproduct of transient TE expression and do not contribute to genome-wide transcriptional changes. Experiments that demonstrate the reduced ability of an organism to overcome stress due to a lack of TE small RNAs are required to assess this theory. Concluding remarks Although bioinformatic approaches have predicted that relationships between TE-derived small RNAs and genic mRNAs are prevalent, to date only two recent publications experimentally verify this mode of regulation. There are many questions that remain, even in these two characterized examples. For instance, the exact mechanism of regulation by TE small RNAs is still unclear, because although one study [23] demonstrated the necessity of the piRNA binding sites in the nos 30 UTR for nos repression, others have shown that these binding sites are not required for rescue of the nos phenotype [68]. It is possible that the TE-derived piRNAs play more of a fine-tuning role in nos mRNA regulation than a Smaug-recruitment role. Additionally, another study [25] reported that the TE-derived siRNA854 could regulate UBP1b mRNA through translational inhibition in one tissue and through transcript cleavage in another. Again, the exact mechanism of regulation in this instance is not clear. It is impossible to determine the scope, evolution, and role of this generegulatory mechanism based on two examples. However, it is now apparent that the categorical division between the regulation of genes and the production of TE small RNAs is not as distinct in vivo, and it is likely that characterization of additional examples will follow. These future examples should further reveal the oftenoverlooked influences of TE activity on the genic transcriptome. Acknowledgments The authors thank Robin Wharton for critically reading this manuscript. A.D.M. is supported by a fellowship from The Ohio State University Center for RNA Biology. Research in the Slotkin laboratory is supported by National Science Foundation (NSF) grant MCB-1020499.
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Further reading Malone, C.D. and Hannon, G.J. (2009) Small RNAs as guardians of the genome. Cell 136, 656–668 623