“Guest list” or “Black list”? Heritable Small RNAs as Immunogenic Memories

Abstract

Small RNA-mediated gene silencing plays a pivotal role in genome immunity by recognizing and eliminating viruses and transposons which otherwise may colonize the genome. However, this can be challenging since individual genomic parasites are highly diverse, and employ multiple immune evasion techniques. In this review, I discuss a new theory proposing that the integrity of the germline is maintained by transgenerationally-transmitted RNA “memories” that record ancestral gene expression patterns, and delineate “Self” from “Foreign” sequences. To maintain such recollection two tactics are employed in parallel: “black listing” of invading nucleic acids, and “guest listing” of endogenous genes. Studies in a number of organisms have shown that this memorization is used by the next generation small RNAs to act as “Inherited Vaccines” that ambush invading elements, or as “Inherited Licenses” that grant the transcription of autogenous sequences.

Overview of transgenerational inheritance

Evolution progresses through an interplay between two major forces, selection and variation. Since the formulation of the Modern Synthesis the theory holds that the environment is limited in its contribution to the evolutionary course of action – it affects selection, but not the degree of variation [1,2]. However, recent discoveries in epigenetics suggest that the environment may influence variability as well [1]. If the parental environment is predictive of the conditions that the progeny will face (often for sessile or short-lived organisms), epigenetic responses can change the output of the encoded genetic information in an adaptive manner [3]. For example, the water flea Daphnia changes its normal developmental course and develops a “helmet” that protects it from predators, if it is born to a mother that survived a similar attack [4].

Over the years, controversy surrounded the study of environmentally affected transgenerational inheritance. Today, an improved understanding of epigenetic mechanisms at the molecular level has provided a logical background to explain how, heretically, an adaptive acquired trait could become heritable [5]. Several epigenetic mechanisms could regulate the expression of relevant genes during transgenerational inheritance including DNA methylation, histone modification, and transmission of regulatory RNAs. Moreover, in many organisms, these pathways appear to be interconnected [5–8].

This review will concentrate on recent discoveries that establish a role for small RNAs in transgenerational transmission of acquired defense. Furthermore, exciting examples of RNA-mediated non-Mendelian genetic effects will be discussed, along with their possible implications for the study of evolution and immunology.

RNA-mediated gene silencing and inheritance

Since the original discovery that small RNAs derived from exogenously-provided double stranded RNA (dsRNA) can regulate gene expression via RNA interference (RNAi), additional endogenous reactive small RNAs species have been uncovered (reviewed in [9]). Small RNA species including microRNAs (miRNAs), PIWI Proteins-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), are 20–30 base pair long and categorized by the specific Argonautes with which they associate [10]. These small RNA species engage with multiple RNA-binding proteins to affect the expression of coding genes, transposons, viruses, and aberrant RNAs. Furthermore, these small RNAs possess unique qualities that promote a long-lasting, and even transgenerational influence: their nuclear activity, their ability to become amplified through the action of RNA Dependent RNA Polymerases (RdRPs) and their ability to undergo stabilizing post-transcriptional modifications (Figure. 1 and Text box).

Figure. 1. Mechanisms for amplification, stabilization, and consolidation of RNAi responses.

Several mechanisms allow RNAi responses to be long lasting, and even transgenerational. (1) viRNAs, and other endogenous small RNA species, give rise to small RNAs which are, in a number of organisms, amplified by RdRPs. The amplified response can produce small RNAs that are 100 fold more abundant than the original trigger small RNAs. piRNAs are amplified by RdRPs in worms, and by a different mechanism termed “Ping Pong” in flies and mice. In addition to amplification, different small RNAs are modified, for example by the small RNA 2’-O-methyltransferase HEN1. Such modifications stabilize the RNA molecule by rendering it inaccessible to nucleases. (2) Apart from cell-autonomous functions, small RNAs move systemically between different cells and tissues. Specifically, small RNAs transfer from the soma to the germline. (3) Aside from post-transcriptional silencing, and “slicing” of mRNA targets, small RNAs work also at the transcriptional level, by inducing chromatin remodeling and DNA methylation, and also by inhibiting Pol II progression. Once established, such epigenetic marks act to recruit the RNAi machinery and thus perpetuate the silencing via a “Feed forward loop”.

Text Box. 1. Mechanisms that enable long-lasting RNAi.

In various organisms dsRNA is identified by RNA-binding proteins, and chopped up by DICER to produce small RNAs [70–73]. After the association with Argonaute proteins, one strand of the short dsRNA intermediate is selected [74,75] to guide the RNA-induced silencing complex (RISC) towards complementary mRNA targets. This cascade ultimately leads to the inactivation of the targeted gene [76]. In certain cases, genes become stably silenced. The establishment of stable silencing was originally thought to be a post-transcriptional process, however, multiple lines of evidence from different organisms suggest that small RNAs also promote silencing in the nucleus at the transcriptional level. It has been shown that small RNAs guide chromatin remodeling proteins to specific DNA sequences by binding to nascent RNA transcripts, leading to cytosine methylation (in plants, fission yeast, and even in mice [8,77]) and deposition of histone modifications (in plants, fission yeast, worms, and flies [78,79]). The interaction between small RNAs and chromatin remodeling factors is reciprocal. For example, heterochromatin is highly enriched for histone H3 lysine 9 trimethylation (H3K9me3), which is known to be involved in transcriptional gene silencing [80]. In Schizosaccharomyces pombe, not only can RNAi direct deposition of H3K9me3 marks on specific gene targets, the deposited histone mark can also facilitate new rounds of RNAi machinery recruitment, thus engaging a sustainable and self-reinforcing feed-forward loop [81,82]. The association of small RNAs with chromatin produces a lasting effect that persists throughout cell division and even across generations.

Another mechanism that produces lasting, and even transgenerational silencing in nematodes, plants and fungi, is small RNA amplification. By using the target RNA as a template, RNA-dependent RNA polymerases catalyze the biogenesis of secondary small RNAs [83]. siRNAs and piRNAs trigger such amplification, a process that generates a secondary RNAi response that persists long after the original RNA transcript is degraded [84–87].

Since RNA molecules are sensitive to nucleases, they need to be actively protected and so in order to persist small RNAs. One such stabilizing process is methylation of the 3′ end of small RNAs. In a number of organisms, the 2’-O-methyltransferase (known as HEN1) methylates the 3’ end of certain small RNA species to increase the stability of the transcript by rendering it inaccessible to nucleases [88,89].

In summary, several mechanisms exist to amplify and stabilize RNAi responses, which enable persisted small RNA-mediated regulation, that lasts not only throughout the lifetime of the organism in which they were initiated, but also in multiple subsequent generations.

Another incredible quality of small RNAs that expands their potential to serve as transgenerational carriers of somatically-acquired traits, is the movement of certain regulatory RNA molecules between different cells and tissues [11]. In C.elegans, RNAi is maintained in the next generation of worms that are fed bacteria expressing dsRNA [12]. Thus, silencing travels from the site of injection to other tissues, including the germline. This observation challenges an important principle in evolutionary biology known as the “Weismann Barrier” (also termed “The second law of biology”), according to which genetic information cannot pass from the soma to the germline [13]. Recently it was demonstrated that higher organisms also use RNAi to silence genes in a non-cell autonomous manner [14,15], suggesting that soma-germline RNA-mediated communication could be widely conserved.

While in most cases wild-type gene activity is restored to baseline after dsRNA exposure in the second generation, targeting certain genes can result in lasting interference [16]. Several factors influence the persistency of RNAi inheritance, and segregation of the effect does not follow Mendel’s rules of heredity - I will now briefly describe the dynamics of this unusual genetic process.

In C.elegans RNAi is inherited as a dominant factor [16] in both the oocyte and the sperm, and the silencing can be more persistent when transmitted through the male germline [17]. This is suppressing since the sperm’s volume is less than 1% of the volume of oocyte. It was hypothesized that the sperm carries large amounts of RNA packed in its perinuclear “halo” [17]. Interestingly, in worms distinct endo-siRNAs are inherited from the father and the mother [18].

In plants, RNAi acts both post and pre-transcriptionally. For example, RNAi can result in heritable DNA methylation, a process that is dependent on the Met1 methyltransferase [19]. Stable RNAi-mediated methylation of DNA cannot explain inheritance seen in worms, since in C.elegans cytosine methylation does not seem to occur [20] suggesting alternative RNAi inheritance mechanisms are utilized as well. Indeed, unlike epigenetic effects which are mediated by DNA modifications, heritable RNAi effects were shown to propagate to the next generation of worms as a diffusible epigenetic element (demonstrated using animals in which deletions were introduced to remove the target DNA), suggesting that in C.elegans the chromosomal locus is either redundant or dispensable for transmission of transgenerational memory [16,17,21]. Recent evidence in worms suggests, however, that RNAi inheritance can involve a nuclear RNAi mechanism and chromatin remodeling genes as well [22]. RNAi inheritance maintained by chromatin remodeling factors (HDA-4, K03D10.3, ISW-1 and MRG-1 [22]) was shown to persist almost indefinitely (>80 generations) in the absence of the original trigger [22]. However, this persistent RNAi-dependent effect was only seen in a small minority of genes (13 out of 171 tested genes were inheritably silenced) [22] suggesting that, for reasons which currently unknown, certain genes are more capable of inheriting RNAi over others. While chromatin remodeling appears to play an important role in epigenetic inheritance it appears that the heterochromatin state is not directly inherited but reestablished in the progeny by small RNAs [23]. This was shown to be the case in worms by Burton et al., who found that although treatment of worms with dsRNA induces heritable deposition of repressive chromatin marks in the progeny, these heritable siRNAs are detectable before the chromatin marks [23]

How can heritable small RNAs persist over many generations? Each nematode produces hundreds of progeny, and therefore small RNAs must be amplified to overcome the transgenerational dilution effect that expands logarithmically. Indeed, in worms which are mutants for the RdRP RRF-1, inherited RNAi diminishes after 2 generations, indicating the RNA amplification is required for long-term transgenerational RNAi effects [21,24]. In addition, it was recently demonstrated that the RRF-1 RdRP is required for effective transgenerational targeting of chromatin [25]. The inherited effect of RNAi on chromatin was shown to be mediated by HRDE-1 (heritable RNAi defective-1, also named WAGO-9; for Worm Specific Argonaute 9) and Nuclear RNAi factors (NRDEs, for Nuclear RNAi Defective). HRDE-1 is a germline-expressed Argonaute protein that binds secondary siRNAs, and while it is required for efficient RNAi inheritance, it is dispensable for RNAi per se [26–28]. HRDE-1 and NRDE proteins probably function in consolidating transgenerational RNAi “memory”, rather than in the actual shuttling of the heritable RNA agent across generations. Indeed, HRDE-1 and NRDE mutant parents are still capable of transmitting RNAi to their progeny, while mutant progeny is incapable of establishing RNAi using the inherited small RNAs received from wild-type parents [23,27,29]. Moreover, HRDE-1 is found in oocytes nuclei but not in differentiated sperm nuclei [27], although RNAi is most efficiently transmitted via sperm [17]. In summary, HRDE-1 and NRDE proteins are important for consolidation of the inherited response, which is triggered by small RNAs, and established by chromatin remodeling [6,27].

Inherited immunity

The active nature of transgenerational small RNA transmission, and the discovery of an elaborate set of proteins which are dedicated to RNAi inheritance but not to RNAi per se, suggests that inherited RNAi was not evolved solely to allow artificial gene silencing of multiple generations in the lab. Thus, the question remains – what is the biological purpose of small RNA inheritance? Recent discoveries provide a clue: in multiple organisms, including bacteria, plants, and animals, inherited small RNAs establish the foundation for transgenerational genome immunity.

Inherited anti-viral immunity

A link between RNAi and anti-viral defense was recently discerned in C. elegans, which was considered for many years to be resistant to all known viruses [30]. RNAi mutants, but not wild type worms, were shown to be susceptible to artificial viral replication [31–33], and recently RNAi-dependency was proven essential for resistance against a natural virus (the Orsay virus) as well [34]. Small RNAs targeting viral genomes (viRNA) accumulate in wild type infected worms, but not mutant worms [35,36]. Moreover, it was shown that viRNAs are capable of being transmitted between generations of worms [21,24]. RNAi heterozygous mutant worms, which are functionally competent, can generate anti-viral RNAi immunity that protects their homozygous RNAi-mutant progeny from replication of a transgenic Flock House Virus (FHV) for multiple generations [21]. Recent experiments suggest that C.elegans worms which are infected with the natural Orsay virus also develop RNAi-based protection which is accumulated over generations, so that the progeny of the infected worms is essentially immuned (Personal communication [37]). Thus, an acquired trait, anti-viral immunity, is transferred between generations via a non-Mendelian genetic mechanism, and in direct defiance of Weismann’s law [21].

Inherited protection against transposons

piRNA-mediated transposon regulation provides interesting examples for transgenerational immunity and for genetic interactions between the soma and the germline. For example, while piRNAs act mostly in germ cells, in Drosophila, somatic piRNAs eliminate a particular transposon even prior to colonization of the reproductive organs. In somatic follicle cells, antisense piRNAs are produced from a Flamenco locus to repress gypsy elements (long terminal repeat reteroviruses), which are able to travel from the soma in viral particles to the germ cells. By blocking selfish genes even before they get to the germline, piRNA-mediated repression might prevent transgenerational mobilization and evolution of these and other mobile elements [38]. piRNAs are also maternally-inherited in Drosophila. This maternal deposition is important for protection of the F1 generation against transposons (and hybrid dysgenesis) [39]. Unlike maternally-inherited proteins, which dilute quickly over generations, piRNAs are amplifiable, and the maternally-deposited piRNAs in flies persist long into adulthood [39]. Moreover, amplification mechanisms allow piRNAs to establish multigenerational defense against a variety of selfish elements [26,28,40,41]. piRNA amplification is achieved by different mechanisms in different organisms. In C.elegans piRNAs give rise to secondary endo-siRNAs via the action of RdRPs [42]. In flies and mice piRNA-mediated silencing is amplified by a different process termed “Ping-Pong” [43].

Long-term piRNA inheritance appears to be well conserved: recently it was shown that piRNAs are inherited for 50 generations in Drosophila [40], and for at least two generations in Danio rerio [44]. While piRNAs are involved in many inherited immunity effects, they are obviously not central to all such mechanisms. piRNAs are absent in several organisms that display heritable genome immunity, such as plants, fungi, and even in some worms, for example in the parasitic nematode, Ascaris suum, a relative of C.elegans [45,46].

Manipulation of transgenerational immunity

Small RNA-based surveillance cannot eliminate all the genetic parasites, since some mobile elements utilize different techniques to overcome, or even manipulate RNAi, to escape inherited immunity. Viruses encode a variety of proteins named Viral Suppressors of RNA Silencing (VSRs) to block the host’s RNAi system [47]. In plants, a class 1 RNase III VSR from sweet potato chlorotic stunt virus inhibits anti-viral RNAi by disabling siRNA amplification by the RdRP RDR6 [48]. Similarly, the Tomato yellow leaf curl virus VSR V2 outcompetes the SUPPRESSOR OF GENE SILENCING 3 (SGS3) protein for binding to the 5’ overhang of dsRNAs, thus preventing its interaction which is required for initiating RdRP-mediated amplification of anti-viral viRNAs [49]. Inhibition of host RdRPs should, in theory, prevent anti-viral RNAi from persisting for long-periods of time, and from acting transgenerationally.

While many examples for transgenerational protection against viruses exist, more notably in plants [3,50], certain epigenetic effects are found that “favor” invading viruses and establish an inherited susceptibility. For example, seeds from injured ancestors were more susceptible to infections by a plant virus [51]. Specifically, some of the signaling cascades that viruses hijack are used by plants to transgenerationally regulate endogenous genes. In A. thaliana, the same SGS3 protein that is outcompeted by the viral protein V2 for dsRNA binding is inhibited by temperature increase. Plants naturally use this mechanism to accustom themselves quickly to changes in temperature: Since SGS3 is required for siRNA production and the ensuing transgenerational post-transcriptional gene-silencing program that they enforce, it was shown that through inhibition of SGS3 temperature-shifts inhibit siRNAs from affecting the next generation [52]. In addition to suppressing RNAi in worms and plants, even in mammalian systems, it was shown that certain viruses hijack the host’s RNAi system to directly silence endogenous immunity genes, and achieve immunoevasion [53].

Virus-host interactions are moreover complicated by situations of persistent infections, when the host can sometimes benefit from “living with the (viral) enemy”, and can even gain from internalization of RNA viruses in to the genome, using the cell’s own reverse transcription abilities [54]. While the underlying mechanisms are unknown, it was discovered that the human genome contains bits and pieces of non-retrovirus RNA viruses [55,56]. A clue to the mechanism might be found in Drosophila, where in cells such integration was shown to be achieved by the reverse transcriptase activity of transposons, resulting in establishment of an anti-viral RNAi response [57]. Therefore, the effectivity of small RNAs-based immunity changes for different mobile elements, from host to host, and from environment to environment, as is evident by the relative genome fraction that the different genomic parasites take up in different organisms [58].

In addition to inherited viRNAs and piRNAs, other inherited small RNA species enable transgenerational immunity not only against viruses and transposons, but also, at least in plants, against animal predators. Small RNAs were demonstrated to mediate resistance in Wild radish (Raphanus raphanistrum) against lepidopteran herbivory that is prolonged for at least two generations [51]. This biotic attack is countered by the production of small RNAs, which spread to the developing seed and establish stable epigenetic protection. Unlike the situation in C.elegans, inherited siRNA-mediated protection in plants is hypothesized to act also by the methylation of specific cytosines, which persist over generations [19]. In summary, it appears that inherited small RNAs are involved in multiple regulatory pathways central to immunity, across the tree of life.

Strategies for establishing transgenerational genome immunity

In general, there are two ways to use prior infections to protect the progeny: the organism can either make a heritable “black list” of all the foreign genes that its ancestors encountered, which must be suppressed, or prepare a “guest list” of all the familial self genes that need be to expressed. Evidences for the existence of both strategies are detailed below.

Black list: Inherited Vaccines

An important strategy for identifying parasitic elements is to take advantage of the virus’s or the transposons’s need to replicate and mobilize between cells or between different parts of the genome [43]. In Drosophila germ cells specific genomic loci act as “traps” catching transposons that land in them when the mobile element jumps. Once the transposon is captured, its expression is restricted, since the DNA is wrapped up by heterochromatin. piRNAs that are synthesized from the transposon’s landing site continuously recruit chromatin remodeling genes to the site. This allows the heterochromatin state to remain permanent after both mitotic and meiotic cell divisions. Also, once piRNAs have been synthesized, they can prevent expression of homologous genetic parasites [43]. Since piRNAs are amplifiable, this transposon memorization is transgenerational. While anti-transposon piRNAs are produced almost exclusively in germ cells, where transposons mobilization is especially deleterious, in A. thaliana 21 bp siRNAs raised against transposons in the germline supporting companion cells function non-cell autonomously to silence transposons in the sperm [59].

In addition to mobilization, the repetitive nature of transposons leads to bidirectional transcription and therefore to dsRNA formation. For non-retrovirus RNA viruses, synthesis of dsRNA intermediates is a prerequisite for reproduction. Therefore, the immune system surveys dsRNA, since it is a typical product of parasitic elements, and possibly a “danger” signal [60]. The dsRNA, which transposons and RNA viruses produce, serves as a substrate for the RNAi machinery, leading to the production of heritable inhibitory viRNA and piRNA “memories” [21,43,60,61], as discussed above.

An analogous identification mechanism also exists in bacteria. Invading DNA phages can be chopped up and packed into specific genomic areas called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) where they can be silenced, and their genomes “memorized” [62]. In subsequent bacterial generations, CRISPR RNAs transcribed from these CRISPR loci will prime the bacteria’s defense against newly infecting phages, thus leading to their elimination [63].

Transposons “trapping”, viRNA inheritance, and phage integration in CRISPR loci, are mechanisms in which regulatory RNAs participate to help establish what I term “Inherited Vaccination”, a transgenerational “Black List” of genetic parasites that need to be eliminated (Figure. 2). The term “Vaccine” might seem unfitting at first since the process does not involve production of antibodies or clonal populations of specialized immune cells, however, functionally, small RNA-mediated inherited immunity is indeed a form of vaccination.

Figure. 2. Regulatory small RNAs are derived from different genomic pathogens, across the tree of life.

Viruses, transposons, and invading phages are all controlled by a variety of regulatory RNA species. The scheme demonstrates how, in different organisms (both eukaryotes and prokaryotes), analogous mechanisms act to eliminate or silence in a heritable manner pathogenic elements by use of RNA molecules, which recruit effector proteins. (1) RNA binding proteins, which engage DICER proteins, recognize viruses that produce dsRNA during the course of replication. DICER processes the viral RNA into small RNA (viRNAs), which are loaded onto Argonaute proteins. In worms the viRNA response is further amplified by RdRPs to create abundant secondary viRNAs. Transposons that land in “traps” give rise to piRNAs. Such piRNAs are amplified by the “Ping Pong” mechanism or by RdRPs (depending on the organism), and ultimately lead to the destruction of the transposon, which is inhibited from expressing. (2) Phages that invade bacteria are restricted and integrated into repetitive CRISPR loci. CRISPR RNAs are transcribed from the CRIPER loci, loaded onto effector complexes and lead to the destruction of similar phages. The immunity that is provided by all of these mechanisms persists for multiple generations.

Guest List: RNA Licensing

The second strategy that an organism can use to produce inherited immunity could be viewed as the mirror image of the first strategy – make a small RNA-based “Guest List” of endogenous genes, instead of a “black list” of invading elements.

The idea that an organism produces an RNA-encoded, heritable list of all helpful genetic information (“RNA Cache”) was validated both in unicellular Oxytricha trifallax, and in animals [64].

Oxytricha ciliates have two genomes packed inside two nuclei, a somatic one that encodes for all the vegetative growth functions, and a germline one that is responsible for sexual reproduction [64]. During the development of the somatic nucleus, Oxytricha gets rid of all parasitic DNA elements, which compose the majority of the germline DNA (>95%). It was known that RNA guides DNA rearrangements in Oxytricha [65], however only recently it was shown that piRNAs are central to this process [66]. piRNAs that match genes present in the somatic genome are transgenerationally inherited from the maternal nucleus. Since these piRNAs correspond to genes that were previously present in the soma, they in fact point to genes that are “safe” to express, because the prior generations that expressed those genes were able to survive and produce progeny. Thus, Oxytricha’s piRNAs can recognize “self” (and “safe”) genes, and retain these corresponding DNA sequences (<5%) in later generations. This model w as validated when synthesized piRNAs that match regions that are otherwise deleted were injected into Oxytricha cells. It was shown that these artificially-produced piRNAs instruct the germline to refrain from deleting the corresponding DNA sequences, so that these DNA regions are kept in later generations [66]. Thus, if RNAi-mediated immunity is normally thought of as a strategy to dispose of “foreign” sequences, the Oxytricha’s RNAi system does the opposite – it protects its genome by discarding its entire somatic DNA, and licensing the inclusion of self-genes only.

The “RNA cache” idea was dubbed later “RNA licensing”, when it was demonstrated in C.elegans that an RNA transcript of the gene fem-1, which is a sex determining factor, needs to be transcribed in the mother, to “license” the expression of the fem-1 allele in the zygotic germline._Introduction of a fem-1 deletion which eliminated mRNA production in the mother resulted in the production of feminized germline in the heterozygous progeny [67]. Similarly to the experiments done in Oxytricha, injection of fem-1 RNA (even when the RNA was incapable of encoding for a protein) into the maternal germline was enough to rescue the deficiency in the offspring. The presence or absence of the maternal RNA produced a heritable effect, which suggested the maternal fem-1 RNA prevents epigenetic silencing in the next generations, and that the underling mechanism may be a way to protect the identity and integrity of the germline [67].

Based on recent studies in C.elegans, it seems reasonable to speculate that the RNA agents that mediate such epigenetic licensing are small RNAs. A number of groups have now shown that heritable worm PRG-1-bound piRNAs (a worm PIWI protein) scan and silence foreign elements by inducing chromatin remodeling in the germline [26–28,41]. One group suggested that small RNAs, which are bound by different Argonaute proteins, serve as epigenetic memories of “self” and “nonself” RNAs. piRNAs, according to this theory, silence the expression of genes which are not already marked by endo-siRNAs (bound by the Argonaute CSR-1), in a heritable manner that persists for multiple generations [26]. piRNAs arise in C.elegans primarily from two clusters on chromosome IV, but nevertheless, according to this model, possess the capacity to downregulate the expression of every foreign genetic element, regardless of sequence composition. The theory holds that piRNAs are granted with such incredible diversity since they require only partial complementary to their targets. Since the worm’s piRNA pool has a limited content to cover every imaginable foreign sequence, these piRNAs (which are only 21 bp long) must be very promiscuous, requiring only ~80% identity for target binding [26,28].

In summary, it is likely that different organisms protect their genomes by utilizing specialized small RNA-based mechanisms that enable both “Black” and “Guest” listing strategies.

Concluding remarks

Earlier in the 20^th century it was suggested that immunity could be heritable [68]. However, the heritable effects that were documented in the past remain extremely controversial, especially due to the fact that no plausible transmission mechanism was suggested. On the contrary, the discoveries of genome protective and transgenerationally transmitted small RNAs stand on solid ground - previous research efforts have unequivocally demonstrated that small RNAs are heritable, and provided insights into the different mechanisms that contribute to the persistency of RNAi.

While a myriad of examples exists for the involvement of inherited small RNAs in defending the genome from threats that come from the “outside” (genomic parasites), it is very possible that transgenerationally-transmitted regulatory RNAs play other “in house” roles as well. Since small RNA could be transcribed in response to different environmental challenges, one hypothesis is that heritable RNA, originally evolved to provide immunity, further allows organisms to tune down the levels of endogenous genes that are harmful for growth or reproduction in a changing surroundings [21,23]. For example, it was recently shown that the bacterial immune system, CRISPR, is also utilized to regulate endogenous bacterial genes [69]. Thus it is possible that the principles that enable heritable acquired immune resistance contribute to the evolution of many additional traits.

Glossary

Selection

The mechanism that allows organisms with genotypes that fit specific environments to survive, reproduce, and increase in number over generations.

Variation

The differences between individuals of the same specie.

Mendelian VS. Non-Mendelian traits

Categoric traits that are regulated by a single locus, and segregate according to Mendel’s laws of inheritance.

The Modern Synthesis

The unification of Darwin’s mechanism of natural selection with Mendel’s theory of heredity, and the principles of population genetics.

Epienetics

Heritable changes that arise independently of changes to the DNA sequence.

Epigenetic transgenerational Inheritance

Heritable epigenetic changes that persist across generations, as opposed to epigenetic changes that are maintained over cell divisions.

Cytosine methylation

The biochemical process involving the addition of a methyl group to the 5 position of the cytosine pyrimidine ring. This modification stably alters the expression of genes, and can be heritable.

Histone modification

Histones can be covalently modified at several sites. There are many different histone modifications including methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination, and ADP-ribosylation. The different modifications comprise a “histone code” that affects multiple functions such as gene expression and chromatin condensation.

Transposons

Are one of multiple species of mobile genetic elements, which change their location in the genome by “jumping”. Transposons are classified based on the mechanism of transposition, which can entail mobilization following self-replication (class I) or by “cutting and pasting” (class II).

RdRPs

RNA-dependent RNA polymerases are enzymes which replicate RNA using RNA as a template.

viRNAs

Small RNAs that correspond to a viral genome, and start a cascade that leads to the elimination of the virus. Typically, primary viRNAs are DICER products that are chopped directly from a dsRNA viral sequence (synthesis of dsRNA intermediates is required for replication of RNA viruses). Primary viRNAs serve as primers for the amplification of secondary viRNAs by RdRPs.

piRNAs

Small RNAs that interact with PIWI proteins. piRNAs are mostly expressed in germ cells, where they silence retrotransposons and other mobile elements.

endo-siRNAs

Autonomously generated siRNAs. The biogenesis of endo-siRNA is still poorly understood, however endo-siRNAs were demonstrated to derive from repetitive sequences and from areas that encode for dsRNA structures, such as sites of convergent transcription. In addition, anti-sense endo-siRNAs can be transcribed off natural mRNAs, presumably via RdRP activity.

Vaccine

An agent that stimulates the immune system to respond against specific pathogens, and typically resembles or is derived from the disease agent itself. For example, vaccines can be made from a weakened virus or bacteria, and can lead to production of antibodies.

Inherited immunity

Immune responses that persist across generations. In the case of heritable small RNAs-mediated immunity, the “vaccines”, siRNAs, viRNAs or piRNAs, are directly derived from the subdued genomic parasite, and target the disease, similarly to antibodies, when the pathogen is next encountered.