Tag Archives: RNAi

Interacting small RNA pathways in worms 5: Global Genome Surveillance

As discussed previously, in C. elegans more than 15,000 21U-RNAs are expressed from two large clusters on chromosome IV. As very few of these piRNAs exhibit perfect sequence complementarity with other endogenous sequences within in the C. elegans genome, it’s been difficult to deduce the targets and functions of this system. New studies from the labs of Eric Miska and Craig Mello have significantly advanced our understandings of piRNAs in C. elegans. Bagijn et al. and Lee et al. have confirmed a previous supposition that 21U-RNAs can base-pair with imperfectly complementary sequences. This less stringent base-pairing opens up the entire transcriptome to possible piRNA-mediated regulation. Bagijn et al, Ashe et al, and Shirayama et al. have further characterised the effector pathways downstream of PRG-1/piRNA targeting. Together these papers outline a finely tuned genomic surveillance mechanism capable of discerning self and non-self transcripts.


Previous studies had shown that 21U-RNAs act upstream of 22G-RNAs to repress the activity of Tc3 transposons. Lee et al. therefore investigated whether the expression of 22G-RNAs was altered in worms mutant for the worm Piwi protein PRG-1. When all 22G-RNAs were considered together, they couldn’t observe a correlation between PRG-1 activity and 22G-RNAs. However, 22G-RNAs can be divided into four different pathways defined by the different AGOs with which they complex. When those associated with 2 different ‘Eri’ pathways, and the CSR-1 22G-RNAs were discounted, Lee et al. observed that WAGO-associated 22G-RNAs tended to be depleted in prg-1 mutants. mRNAs targeted by WAGO 22G-RNAs showed a tendency towards upregulation in prg-1 mutants, consistent with a repressive role for these small RNAs.

22G-RNAs are not evenly distributed on their target mRNAs. Instead, hotspots occur where 22G-RNA species are far more common than at other points on the same mRNA. Lee et al. postulated that these hotspots could be regions where PRG-1/21U-RNA complexes recruit RNA-dependent RNA polymerases to generate 22G-RNAs. If this is as widespread a phenomenon as suggested by the depletion of 22G-RNAs in prg-1 mutants, imperfect base-pairing of 21U-RNAs to their targets must be occurring, as only 29 WAGO targets show perfect complementarity to 21U-RNAs. Lee et al. therefore asked whether 22G-RNAs are enriched in mRNA coding regions with the potential for energetically favourable but imperfect base pairing to 21U-RNAs. Basing their parameters on the imperfect base-pairing observed for miRNA target interactions (in which strong base-pairing in a ‘seed’ region of nucleotides 2-8, facilitates less perfect pairing between the rest of the sequences), Lee et al. determined the level of 22G-RNAs within a 100nt window around potential 21U-RNA binding sites in wild-type and prg-1 mutant worms.  Allowing 2 mismatches and 1 G:U wobble pair outside the seed region, and at most 1 G:U pair within the seed (parameters that meant that more than 50% of genes contained potential 21U-RNA binding sites), the researchers found that 22G-RNAs mapping to within ± 50nt of the 21U-RNA binding sites on WAGO mRNA targets, were 3 fold enriched in wildtype worms relative to the prg-1 mutants. A smaller enrichment of 1.4 fold was also observed for CSR-1 mRNA targets. The level of 22G-RNA enrichment correlated with the expression levels of 21U-RNAs. Lee et al. confirmed the importance of pairing within the seed region by comparing this data with another set of potential 21U-RNA targets with similar total mismatches but poor seed pairing, which showed little 22G-RNA enrichment.

Bagijn et al performed slightly different analyses, which came to similar conclusions. They identified 681,746 potential binding sites for 16,003 21U-RNAs if 3 mismatches are allowed (not biased away from seed region). They then looked to see what proportions of these sites 22G-RNAs also mapped to, finding 1.6, 1.4, 1.5 and 1.2 fold 22G-RNA enrichments relative to matched control sequences, at 0, 1, 2 and 3 mismatch sites respectively. As with Lee et al. the levels of 22G-RNAs correlated with 21U-RNA abundance.

These analyses suggest that 21U-RNAs are targeting a large proportion of the germline transcriptome, including both protein-coding genes and transposons. In relation to the repression of Tc3 (discussed in the previous post), Bagijn et al. identified three piRNAs with imperfect complementarity to sequences in the TIRs, perhaps clearing up some of the ambiguities from previous studies. By using their data on 22G-RNA enrichments at potential 21U-RNA binding sites, they were able to rank the likelihood of piRNA regulation for transcripts. Six of eleven candidates that showed strong reductions in 22G-RNAs in prg-1 mutants also showed statistically significant transcriptional upregulation (including both transposons and protein-coding genes).

piRNA sensors

Most of the papers under discussion used transgenic ‘sensor’ lines to dissect the mechanisms of PRG-1/21U-RNA mediated silencing. Bagijn et al generated a ‘piRNA sensor’ in which a GFP – histone H2B fusion gene with a sequence complementary to a known 21U-RNA is inserted into the C. elegans genome. In the control sensor line (in which the reverse complement of the 21U-RNA is included instead), the GFP reporter protein is expressed in germline nuclei. In the piRNA sensor line the transgene is silenced. This silencing is dependent on PRG-1, as the sensor was desilenced in prg-1 mutants. Bagijn et al detected 22G-RNAs mapping to the piRNA sensor mRNA close to the piRNA target site, that weren’t present in the control sensor or in prg-1 mutants. As levels of both the piRNA sensor pre-mRNA and mRNA were raised in prg-1 mutants, it appears that silencing occurs at the level of transcription, and possibly post-transcriptionally as well.

By crossing the piRNA sensor line into various mutant worm strains, Bagijn et al showed that the silencing involved many of the 22G-RNA pathway components discussed previously, including the helicase DRH-3, the RdRPs EGO-1 and RRF-1, and various WAGOs. As has been discussed previously, many of the WAGO proteins have overlapping functions and hence display partial redundancy when impaired. However, wago-9 (also known as hrde-1) appears to be an especially important AGO in the silencing process; the sole single WAGO mutant to cause desilencing phenotypes. All these 22G-RNA components act downstream of PRG-1, as 21U-RNAs are still present in these mutants while 22G-RNAs fail to accumulate. In prg-1 mutants both classes of small RNAs are affected.

Although in other animals Piwi proteins are known to act by slicing it’s targets, transgenes encoding PRG-1 with a mutated endonuclease motif could rescue the desilencing observed in prg-1 mutants. By mutating the piRNA target sequence in the sensor, Bagijn et al. then showed that 2 mismatches are tolerated for piRNA mediated silencing to occur. The residues changed could be anywhere in the sequence including at positions 10 and 11 – the normal site for Piwi slicing activity.

Lee et al. undertook very similar experiments to Bagijn et al, generating a piRNA sensor line and confirming that mismatches with the piRNA target site were tolerated, including at the normal slicing site. However there is a crucial difference between the two sets of experiments using piRNA sensors. In Bagijn et al’s study, PRG-1 was required continuously for silencing to be maintained through the generations. That is to say that an already silenced sensor would be desilenced when crossed into a prg-1 mutant line. Lee et al’s piRNA sensor only required PRG-1 for silencing to be initiated, but not for it to be maintained. When Lee et al’s silenced sensor was outcrossed into prg-1 mutants, GFP expression was not activated, whereas if the transgene was introduced into prg-1 mutants it was not silenced. This important disparity may well be caused by differences in the (broadly similar) compositions of the sensor transgenes, and will be discussed later.

Long-term heritability.

The PRG-1-dependent silencing of the piRNA sensors is incredibly long lived, being transmissable through many generations. This type of epigenetically heritable effect has been previously observed with various RNAi paradigms in C. elegans, with disagreement over whether epigenetic transmission is primarily through inheritance of small RNAs, or via chromatin modifications, or both (see these earlier posts: 1, 2 ). Ashe et al. generated another transgenic sensor line to monitor the heritability of dsRNA-induced RNAi. They found that both the piRNA-mediated silencing pathway and the heritable RNAi silencing pathway converge on a common group of nuclear factors responsible for 22G-RNA mediated silencing. These include two proteins of unknown function, NRDE-1 and NRDE-2, known to mediate nuclear RNAi via interactions with nascent transcripts; the heterochromatin protein 1 orthologue, HPL-2; the nuclear AGO WAGO-9; and a SET-domain protein, SET-25, thought to be a methyltransferase responsible for histone H3 lysine-9 trimethylation (a repressive chromatin modification). This collection of downstream effectors suggests that the heritability of both RNAi and piRNA mediated silencing rests on epigenetically stable chromatin marks.

However, if a silenced piRNA sensor strain is crossed with a strain expressing a independent GFP transgene, a dominant silencing of both transgenes occurs. This trans­-acting silencing effect is most likely mediated by secondary siRNAs (ie 22G-RNAs). When small RNA populations are assayed after the induction of RNAi silencing, a large diversity of small RNAs with little 5’ bias (ie Dicer products) were found to target the sensor. However, by the 4th generation, targeting small RNAs had clarified themselves into antisense 22G-RNAs. These 22G-RNAs appeared to be generated de novo in each generation. It therefore appears likely that epigenetic silencing is achieved by the inheritance of both small RNAs and chromatin marks, is reaffirmed in each generation, and acts at both transcriptional and post-transcriptional levels.


In the previously discussed papers, transgenes that were engineered to contain 21U-RNA binding sites were actively silenced in C. elegans. Shirayama et al. describe how transgenes containing non-endogenous genes can be silenced even in the absence of perfect piRNA recognition sequences. They found that when transgene fusions of gfp  and endogenous genes were inserted into the genome, they were occasionally completely silenced, whilst sometimes exactly the same construct, inserted into the same genomic site, was expressed. When a silent line was crossed to an expressing line, the transgene was invariably silenced in 100% of progeny. This dominant trans-acting silencing was heritable through many generations.  Shirayama et al termed this phenomenon RNA-induced epigenetic silencing (RNAe)(rather forestalling there own explanation of it’s causes).

By analysing levels of pre-mRNAs and mRNAs in silenced and active lines, Shirayama et al. found that silencing occurs at both the transcriptional and post-transcriptional levels. Silenced alleles were activated when crossed into lines mutant for various factors involved in repressive chromatin formation (the polycomb group proteins MES-3 + 4, and heterochromatin protein HPL-2). Whilst ChIP experiments demonstrated that silenced transgenes were enriched for histone H3K9 trimethylation. Hence RNAe involves transcriptional silencing at the level of chromatin formation.

The trans- acting nature of RNAe suggested a small RNA component. Crossing silenced alleles into lines mutant for factors known to function in the WAGO 22G-RNA pathway (rde-3,+mut-7) resulted in desilencing. Similarly transgenes were desilenced in worms mutant for the nuclear AGO encoding wago-9; effects which could be enhanced by mutations in additional wago genes. Sequencing of small RNAs from silenced worm gonads revealed a strong accumulation of 22G-RNAs targeted against gfp. The gfp genes were always combined with endogenous genes in the transgene constructs. Interestingly, 22G-RNAs were not produced against this part of the transgene, suggesting a mechanism protecting against the silencing of endogenous genes.

When a silenced transgene is crossed into prg-1 mutants it does not become reactivated, however when Shirayama et al performed transgenesis directly into prg-1 mutants silencing failed to occur. This demonstrates that PRG-1 is required for the initiation of RNAe and not for it’s maintenance (in agreement with the findings of Ashe et al and Lee et al). Although Shirayama et al did not find the exact piRNA recognition sites triggering this silencing, they did apparently identify a number of candidate piRNAs, the recognition sites of which displayed heightened expression of 22G-RNAs. It seems therefore that the recognition of foreign nucleic acids and their suppression via RNAe is the primary function of the PRG-1/21U-RNA system.


All of these studies are in general agreement about the basic dynamics of piRNA silencing in C. elegans. piRNA target recognition is mismatch tolerant. PRG-1 does not act by endonuclease activity. Instead, upon recognition it recruits a RdRP complex to generate 22G-RNAs against sequence adjacent to the 21U-RNA binding site. These 22G-RNAs complex with WAGOs (especially WAGO-1 and WAGO-9) that effect genetic silencing at both the level of repressive chromatin and post-transcriptionally. PRG-1/21U-RNA recognition triggers a self-sustaining WAGO-22G-RNA dependent silencing.

However, there is still a question about the targets of this system. With a few mismatches tolerated the known repertoire of 21U-RNAs can target the whole genome. Obviously, it’s not all repressed, so just what is happening? The computational analyses of Bagijn et al suggested that protein-coding mRNA sequences showed signs of bias against potential piRNA recognition sequences, whilst transposon and pseudogene sequences did not.  They also found evidence to suggest that transposon insertions between the conserved 21U-RNA promoter motif and the 21U-RNA gene itself generate new transposon-targeting piRNAs. These findings lead to a model of C. elegans piRNA biology with similarities to that observed in Drosophila; piRNA clusters rich in transposon sequence, generating piRNAs primarily used to repress transposon mobilisation in the germline, with endogenous genes being selected to avoid piRNA mediated repression. Lee et al. did find similar trends but were not convinced that the numbers were strong enough, instead emphasising that many of the findings in these experiments suggest the existence of an anti-silencing pathway.


When Shirayama et al. used dsRNA to silence gfp containing transgenes that hadn’t been silenced by RNAe, they were surprised to find the silencing induced was inherited very stably, whereas RNAi against gfp transgenic lines that had been produced by earlier methods never silenced as heritably. They then crossed these earlier transgenic lines to silenced gfp lines to test whether they were susceptible to trans-silencing. Instead they found that these lines would dominantly activate gfp expression. This suggests the presence of a dominant trans-acting mechanism competing against the trans-acting silencing mechanism.

Another line of evidence suggesting the existence of an anti-silencing pathway is the difference in the requirement for prg-1 for silencing between the piRNA sensors of Bagijn et al and Lee et al.  In the Bagijn et al sensor the 21U-RNA target site was flanked by endogenous sequences that are known targets of CSR-1 22G-RNAs. In contrast, in Lee et al’s sensor the piRNA recognition site was surrounded by sequences not targeted by this small RNA population.

A likely explanation for why the silencing of the Bagijn et al sensor required prg-1 in each generation, was that a CSR-1 based anti-silencing pathway counteracts the silencing induced by PRG-1 triggered WAGO mediated silencing. Likewise, CSR-1 22G-RNAs could be the trans-acting anti-silencing agents suggested in Shirayama et al’s experiments. Shirayama et al’s fusion transgenes were more or less susceptible to silencing depending on the endogenous gene fused to gfp, and as noted earlier, in general silencing associated 22G-RNAs were only enriched for the gfp genes and not against the endogenous sequences in the transgenes.

It therefore seems that an anti-silencing pathway exists that licenses ‘self’ transcripts, protecting them from WAGO 22G-RNA silencing. The CSR-1 pathway is the perfect candidate for this activity, as its’ associated 22G-RNAs are known to target thousands of germline expressed transcripts without inducing silencing. Further dissection of these pathways is obviously necessary to prove CSR-1’s role, and whether the anti-silencing pathway also acts downstream of PRG-1, or just antagonistically to it.

The Miska lab agrees about the existence of the licensing pathway, however they suggest that some of the results indicate the existence of another mechanism. Ashe et al. reported that their piRNA sensor could be stably silenced in a prg-1 independent manner, but only if it had been present in a heterozygous state for multiple generations. This is evidence of a mechanism for detecting unpaired chromatin during meiosis. Shirayama et al’s findings too are indicative of a mechanism for detecting unpaired alleles. This could have significant implications evolutionarily, as it may facilitate the phenotypic expression of recessive traits.

One can describe this self and non-self recognition system as a type of acquired genetic immune system. Although, as yet, the details are only understood in outline, it appears to be a exquisitely finely tuned, and effective system. Until recently no viruses were known to infect C. elegans. This system must be part of the reason why. There are still major questions about to what extent transposons are controlled by the piRNA system in C. elegans and how much of its’ functionality is devoted to the regulation of endogenous genes. As is evident from this series of posts, C. elegans has so many different AGOs, and so many different facets to it’s RNAi systems (germline + soma RNAi, nuclear + cytoplasmic RNAi, RNAe etc) that it can be very difficult to dissect them from each other and then be able to see them in the round. Perhaps the most interesting aspect of this work is the light shed on piRNA and RNAi systems in vertebrates and Drosophila. Secondary siRNA systems have not been found in these organisms, so this system of genetic immunity does not work in the same way in these other clades. The 21U-RNA triggering 22G-RNA generation mechanism has been likened to the amplification of secondary piRNAs by the ping-pong amplification system in Drosophila (a system missing in C. elegans). However, the most fascinating aspect of this work could be the light shed on vertebrate piRNA systems. Hundreds of thousands of ‘pachytene’ piRNAs, without known targets, are expressed in the mammalian germline. Do these piRNAs also tolerate mismatches when binding? And do they also mediate a genome surveillance system capable of detecting non-self transcripts? or unmatched chromatin? Are mammalian transcripts marked in some way to avoid this silencing? No doubt these C. elegans studies will invigorate research in mammalian piRNAs, and no doubt I’ll revisit it soon.

See also: Epigenetic Licensing of a Sex Determination Gene
The CSR-1 siRNA pathway gets more enigmatic

Bagijn MP, Goldstein LD, Sapetschnig A, Weick EM, Bouasker S, Lehrbach NJ, Simard MJ, & Miska EA (2012). Function, targets, and evolution of Caenorhabditis elegans piRNAs. Science (New York, N.Y.), 337 (6094), 574-8 PMID: 22700655

Lee HC, Gu W, Shirayama M, Youngman E, Conte D Jr, & Mello CC (2012). C. elegans piRNAs Mediate the Genome-wide Surveillance of Germline Transcripts. Cell, 150 (1), 78-87 PMID: 22738724

Ashe A, Sapetschnig A, Weick EM, Mitchell J, Bagijn MP, Cording AC, Doebley AL, Goldstein LD, Lehrbach NJ, Le Pen J, Pintacuda G, Sakaguchi A, Sarkies P, Ahmed S, & Miska EA (2012). piRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans. Cell, 150 (1), 88-99 PMID: 22738725

Shirayama M, Seth M, Lee HC, Gu W, Ishidate T, Conte D Jr, & Mello CC (2012). piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline. Cell, 150 (1), 65-77 PMID: 22738726

Interacting small RNA pathways in worms 1: Introduction

A cluster of new papers, in the journals Cell and Science, discuss the links between piRNAs and various endogenous siRNA pathways in the nematode worm C. elegans. Emerging from these experiments is a picture of a genome-wide surveillance system capable of differentiating between self and non-self nucleic acids. The commonalities and differences between these papers require rather detailed analyses. I’m therefore intending to write a series of posts; first covering some of the background information on these small RNA systems and then getting onto the new findings.

A panoply of small RNA molecules, involved in diverse cellular functions have been discovered in the wake of the initial observation of RNA interference (RNAi). Originally RNAi described the mechanism by which genes could be specifically silenced by the exogenous application of cognate double-stranded RNAs. Nowadays, the term RNAi is more generally applied to gene silencing pathways involving the three major classes of small RNAs; microRNAs (miRNAs), small-interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs). A common feature of all these small RNAs is that they complex with members of the Argonaute (AGO) family of proteins. Embedded within AGOs, the small RNAs act as guides; base-pairing with specific target RNAs that can then be cleaved by the RNase H endoribonuclease activity of the AGO protein. However, not all argonautes act by this ‘slicing’ activity; gene silencing can also be achieved by interactions with pathways involved in chromatin modification, or the inhibition of transcriptional elongation. Meanwhile the list of non-silencing roles of AGOs and their complexed small RNAs continues to grow; chromosome segregation, double-strand break repair, programmed genomic rearrangement etc.

The synthesis of both miRNAs and siRNAs generally involves the recognition of dsRNA and its cleavage by Dicer enzymes. miRNAs are derived from short stem-loop structures found in transcripts. siRNAs are the main effectors of the ‘classical’ RNAi. Exogenous dsRNA molecules are cleaved by Dicer into 20-30nt siRNAs that are loaded onto AGOs. However, this pathway also targets endogenously formed dsRNA, which can derive from hairpin structures in transcripts, or by base-pairing between transcripts produced either from separate loci, or by bidirectional transcription at individual loci. Hence, endogenous siRNAs generally target transposons or other repetitive sequences, but can target genes as well.

This basic siRNA pathway is present in most animals, but more complicated systems exist in plants, fungi and nematode worms. These ‘secondary siRNA’ systems wrest on the use of RNA dependent RNA polymerases (RdRPs) to amplify siRNAs against targets recognised by primary siRNAs. In the cases of plants and fungi the dsRNAs produced by RdRPs are again processed by Dicer enzymes and then loaded onto AGOs. However, various populations of RdRP-produced siRNAs in C. elegans do not require Dicer cleavage.

As noted above, the key effectors of these small RNA pathways are argonaute proteins. The numbers of AGOs present in organisms varies widely. The Drosophila genome encodes 5 AGOs; 3 of these are involved in the piRNA system, whilst the other two specialise in either miRNAs or siRNAs. In contrast, C. elegans has 27 AGOs. This reflects the presence of various additional networks of endogenous- siRNAs. Deep sequencing in C. elegans has revealed a large diversity of different varieties of small RNAs, with major peaks at 21, 22, and 26 nt. Different types of sRNAs have different biases in relation to their predominant 5′ residue, 3′ modifications, and extent of 5′ phosphorylation.

This series of posts will ignore many classes of C. elegans siRNAs and instead concentrate on two varieties of 22nt endo-siRNAs with 5′ guanosine residues (22G-RNAs). 22G-RNAs associated with the AGO CSR-1 have been shown to play critical roles in chromosome segregation during meiosis and mitosis. Another population of 22G-RNAs that associate with various worm-specific AGOs (WAGOs) have been implicated in the long-term silencing of transposons and other genomic loci. The piRNAs of worms – 21U-RNAs – display some critical differences with those found in Drosophila and mammals. However, understanding their role in C. elegans may help to explain some of the outstanding questions about their functions thoughout animals.

Ketting RF (2011). The many faces of RNAi. Developmental cell, 20 (2), 148-61 PMID: 21316584

See also these related posts:
small silencing RNAs. I: Piwi-interacting RNAs.
RNAi and Chromatin Modification
Lamarckian inheritance of antiviral response in Nematodes.
Double-strand break interacting RNAs (diRNAs)

RNAi and Chromatin Modification

RNAi silences genes by targeting mRNAs for degradation. However, a second mode by which RNAi effects gene silencing has emerged: by triggering chromatin modifications. Gu et al have analysed the pattern of a specific chromatin modification in response to exogenous double stranded RNA (dsRNA) in C. elegans and show that RNAi triggered chromatin modification is target gene specific and transgenerationally heritable.

The ability of exogenous dsRNAs to silence homologous target genes (RNA interference, RNAi) was discovered in the nematode worm, C. elegans, approximately fifteen years ago. Feeding worms bacteria expressing dsRNA, or bathing worms in dsRNA, has the ability to specifically block gene function and most surprisingly this effect in C. elegans is inherited for some generations. RNAi has become an incredibly useful technique in biology as it works to a greater or lesser extent throughout eukarya, and offers a simple and fast method for compromising gene action specifically. Uncovering the mechanisms by which RNAi works has also opened up huge new vistas on cellular function: namely the proliferation of newly identified classes of endogenous RNA molecules and the discovery of their crucial roles in cellular regulation.

In C. elegans the mechanism of RNAi can be divided into two phases: Firstly, dsRNA is cut into 20-30nt molecules (primary short interfering (si) RNAs) by the enzyme Dicer. These siRNAs complexed with Argonaute proteins recognise and target mRNAs for degradation. The second phase (only present in some organisms) is the de novo synthesis of secondary siRNAs by the primary siRNA/Argonaute mediated recruitment of RNA directed RNA polymerases that use the target mRNA as a template. Apart from cytoplasmic siRNA mediated mRNA degradation, RNAi also functions in the nucleus by siRNA/Argonaute complex interactions with nascent mRNAs and RNA polymerase, and by causing chromatin silencing by histone modifications.

Gu et al have used ChIP-seq (Chromatin immunoprecipitation followed by high throughput sequencing) to make genome wide assessments of the effects of RNAi on the extent of a specific histone modification associated with transcriptionally silenced chromatin (histone 3 lysine 9 trimethylation, H3K9me3). RNAi against the gene lin-15B caused an enrichment of H3K9me3 at the lin-15B locus that spread as far as 9kb from the trigger region (the sequence directly targeted by dsRNA) meaning that two neighbouring genes also showed higher H3K9me3 modifications. No other genomic locations showed H3K9me3 enrichment, meaning that RNAi effects on this chromatin modification are specific to the target gene and neighbouring loci. The same pattern was seen when this experiment was repeated with RNAi against three other genes.

RNAi using dsRNA directed at target sequences that are not transcribed into mRNA was unable to affect H3K9me3 levels, showing that interactions with target mRNAs are essential for RNAi triggered chromatin modification. Likewise, RNAi on worm strains defective for various argonaute proteins that are necessary for secondary siRNA biogenesis, failed to trigger H3K9me3 chromatin modifications.

RNAi mediated gene silencing can last for multiple generations in C. elegans, however it is unclear whether these heritable silencing effects are mediated by inherited siRNAs, by a chromatin based mechanism or by a combination of the two.   Gu et al profiled H3K9me3 and small RNAs through three generations after dsRNA exposure. In the first generation of offspring, H3K9me3 enrichment occurred at a similar level as in the parental worms, although the level of siRNAs had fallen off drastically. The chromatin response was still present in the second generation but had fallen away to background levels by the third generation. These results suggest that H3K9me3 chromatin modifications induced by RNAi are transgenerationally inherited without a requirement for inherited siRNAs or trigger RNA, but are not conclusive.

These results are consistent with an emerging model in which secondary siRNA/ argonaute complexes, transported to the nucleus, direct chromatin silencing by interacting with nascent RNAs or with cognate DNA sequences. Histone modification is then propagated some distance from the trigger sequences. The finding with regard to heritability, are inconclusive and seem to be potentially at odds with a previously discussed paper regarding heritable antiviral response in C. elegans. No doubt we’ll be revisiting this subject matter soon.

Gu, S., Pak, J., Guang, S., Maniar, J., Kennedy, S., & Fire, A. (2012). Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint Nature Genetics DOI: 10.1038/ng.1039

Retrotransposons as regulatory elements

In a paper from 2004, Peaston et al reported on the expression of various retrotransposons (RTEs) in the mouse oocyte and pre-implantation embryo, finding widespread RTE transcription and the presence of chimeric transcripts composed of host genes and RTEs.

In a cDNA library constructed from full grown oocyte (FGO) transcripts, 12% of sequences were derived from MT (mouse transcript, a member of the MaLR family of nonautonomous LTR class III retrotransposons), whilst in a library from 2 cell stage embryos, 3% of cDNAs were derived from murine ERV-L (another class III  LTR RTE). Expression of these and other RTEs tailed off to nothing by the blastocyst stage. The differential developmental expression profile of these RTEs is interesting: MT is a large component of the maternally contributed RNA in the oocyte, whilst MuERV-L must be expressed zygotically very early in development.

The most important finding of this paper was that the cDNA libraries from FGO and 2 cell embryos contained many chimeric gene transcripts in which the 5′ sequence was derived from retrotransposons. These chimeric mRNAs made up 3% of the FGO library and 1.4% of the 2 cell stage embryo library. A large variety of RTEs contribute to chimeras in the FGO library but 51% of them involved MT. 56% of chimeric transcripts in the 2 cell stage had 5′ contributions from MuERV-L and it’s relatives, so RTE composition of the chimeric transcripts correlated with specific RTE abundance. The genes expressed as chimeric transcripts didn’t show any particular functional bias.

When the chimeric transcripts were compared with genomic sequence it was found that the cognate RTEs were either located within the gene locus or upstream of it. If the RTE was encoded within the gene, the chimeric transcript lacked any exons upstream of it. When the RTE was located upstream of the gene, the chimeric mRNA often lacked one or more 5′ exons (2/3rds of the time).

Therefore it appears that RTE sequences act as cis-regulatory elements driving oocyte and pre-implantation embryo specific expression of a population of alternatively spliced transcripts encoding (generally) variant proteins. The notion of RTEs as alternative promoters is close to that of transposons as “controlling elements” put forward by their discoverer Barbara McClintock. The authors note that RTE insertions could give rise to co-regulated gene expression and that RTE driven transcription of multiple host genes “provides grounds for selection of new modes of gene regulation by introducing variation”.

In a review of this work Shapiro uses this as evidence for a “functionalist” perspective, in which he regards mobile elements as “distributed genomic control modules”. This does seem to overstate the purposiveness of TE insertion. One potentially forgets all the cases of deleterious mutations leaving no issue. However, there is no doubt that through evolutionary time, host/parasite arms races can become coevolved integrated functions. An interesting finding in Peaston et al was that sense and antisense transcripts were found in relatively equal ratio when MuERV-L was expressed. This suggested that dsRNA would be formed, triggering RNAi that could seed heterochromatin formation to repress RTE expression. This is again open to a dichotomy of interpretation: in that this is part of a host mechanism to inhibit genomic parasites, or conversely (as Shapiro does) “another mechanism by which RTE insertions can influence the expression of nearby coding sequences and act to construct distributed suites of co-ordinately regulated loci”.

See also: On Transposable Elements and Regulatory Evolution 

Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Solter D, & Knowles BB (2004). Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Developmental cell, 7 (4), 597-606 PMID: 15469847

Shapiro JA (2005). Retrotransposons and regulatory suites. BioEssays : news and reviews in molecular, cellular and developmental biology, 27 (2), 122-5 PMID: 15666350

Lamarckian inheritance of antiviral response in Nematodes.

Rechavi, O., Minevich, G., & Hobert, O. (2011). Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans Cell, 147 (6), 1248-1256 DOI: 10.1016/j.cell.2011.10.042

A new paper in Cell shows a non-mendelian multigenerational inheritance of an acquired trait in the nematode Caenorhabditis elegans.

It has been known since the 1990s that the induction of RNA interference (RNAi) by the exogenous application of double stranded RNAs leads to specific gene silencing and that in C. elegans these effects are often inherited by the worms progeny. However, the mechanism of this transmission has remained unclear, as have the potential biological roles. This new study uses a series of elegant genetic crosses and a modified viral reporter transgene to clarify these outstanding questions.

The authors used a transgenic worm that supports the autonomous replication of the single strand RNA nodavirus Flock House virus (FHV) modified to express GFP and make it’s replication inducible by heat shock. In the first series of experiments, worms heterozygous for certain components of the RNAi pathway (rde-1 or rde-4) that also contained the heat inducible viral transgene were generated. Upon induction of viral replication a robust antiviral response occurs meaning that no GFP is expressed. When these worms self fertilise to produce a new generation, a quarter of the offspring are homozygotes for the mutant rde1 or rde4. These worms would be expected to be unable to silence viral replication as their RNAi mechanisms are non-functional. Instead they still do not express GFP after heat shock indicating that viral silencing continues. This silencing effect continues for several generations until it gradually ‘wears off’. However this ‘fading’ mode of silencing only occurred in a subset of the worms, in others a more stable inherited silencing occurred where GFP expression never reoccurred after many generations. When these two types of worms were crossed all the offspring had the viral GFP signal eliminated. This showed that the suppression of viral production was transmitted in a non-mendelian fashion, in accord with the silencing factors being diffusible trans-acting factors (rather than a hypothetical genomic locus suppressing virus production that would have segregated in a mendelian manner).

In another series of genetic crosses the authors went on to show that the transgenerational viral silencing was maintained in the absence of the viral template. Finally, the authors isolated viRNAs complementary to regions of the viral genome from worms that must have inherited them from their grandparents.

This new research importantly shows a physiological context for transgenerational  transmission of RNA mediated gene silencing, ie in inherited antiviral immunity. It also shows that the mechanism of the transmission of gene silencing can be mediated by inherited small RNA molecules.