Tag Archives: 21U-RNA

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.

 Targets

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.

RNAe 

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.

Discussion 

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.

 Anti-Silencing

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 4: 21U-RNAs.

A survey of C. elegans small RNAs from 2006 (Ruby et al) first reported the discovery of a large class of 21nt RNAs with 5’ uridines – 21U-RNAs. The majority of these RNAs mapped to two distinct regions of the C. elegans genome located on chromosome IV. Generally, they were found between genes or in introns, and showed no sense or antisense bias (when within introns). Each 21U-RNA genomic locus shared an upstream sequence motif. This 34bp motif centred on an 8bp core consensus located 38bp upstream from the start of the 21U-coding sequence. Ruby et al had mapped 5,302 unique 21U-RNAs, but using the upstream consensus they identified 10,807 putative 21U-RNA generating loci on chromosome IV. The functional significance of the upstream motifs was not clear; it seemed probable that they could act as promoters, and that 21U-RNAs were individually transcribed, but it was also feasible that these sequences were sites for targeted cleavage of longer transcripts, or that they were recognition sequences for RNA dependent RNA polymerases. Interestingly, the upstream motif was conserved in the related nematode C. briggsae, but none of the 21U-RNAs themselves were. Very few 21U-RNAs matched other sequences in the genome, but in total they encoded a huge diversity of sequence. It appeared that evolutionary pressure was maximising this sequence complexity rather than maintaining sequence identity. The most important questions on 21U-RNAs were therefore clear from the start; what are the targets? What’s the function of the system?

In 2008, parallel studies from the labs of Craig Mello and Eric Miska further characterised 21U-RNAs (Batista et al. Das et al). As well as bringing their total number to 15,722, they discovered that they were solely expressed in the germline and associate with a Piwi-family Argonaute protein, PRG-1. prg-1 null mutants display dramatic reductions in germ cell numbers as well as an independent temperature sensitive fertility defect. 21U-RNAs fail to accumulate in prg-1 mutants, and co-immunoprecipitate with PRG-1. Like many other Piwi proteins, PRG-1 is localised to the perinuclear nuage in the germline (P-granules).

21U-RNAs are therefore the piwi-interacting RNAs (piRNAs) of C. elegans. Like piRNAs in vertebrates and Drosophila, they are 5’ monophosphorylated, have 5’ uridine residues, and are 3’ modified. Most importantly they interact with a Piwi-family AGO, and are implicated in germline functions. However, they do display some major differences not encountered in piRNAs from other clades; they are only 21nt long (whilst those in vertebrates and Drosophila are 24-30nt in length) and most strangely they appear to be individually transcribed. As with 21U-RNAs, piRNAs in other animals are generated from large genomic clusters. However, in Drosophila and mammals individual piRNAs  derive from larger cluster transcripts. Another important difference with other piRNA systems was the lack of a ping-pong piRNA amplification system.

As the main known role for PIWI/piRNA systems is the silencing of transposable elements, Das et al. and Batista et al. looked for evidence of a similar function for the PRG-1/21U-RNA system. Das et al. screened worms mutant for both prg-1 and the closely related gene prg-2 for signs of transposon desilencing. The only transposon found to be affected was Tc3.  Expression of the Tc3 transposase mRNA was higher, and the reversion rate of mutations caused by Tc3 insertions increased 1000 fold in the double mutants (interestingly, the reversion rate was up only 100 fold in prg-1 single mutants; the only evidence of prg-2 having a functional role in the 21U-RNA system). Das et al. identified a single 21U-RNA that mapped to the sense strand of the Tc3 transposase gene. They then found that a large number of endogeneous siRNAs (ie WAGO associated 22G-RNAs) targeted against both the transposase gene and the terminal inverted repeats (TIR) were strongly depleted in the prg1+2 mutants. Batista et al reported slightly different findings: They identified a different 21U-RNA mapping to the TIR of Tc3 in the same orientaion as the transposase gene. When they searched for endo-siRNAs against Tc3 that were depleted in the prg-1 mutants, they only found that those targeted against the TIR were affected.  The discrepancies between the two studies could indicate slightly different roles for the two PRG proteins.

Importantly, both studies found that the PRG-1/21U-RNA system functions upstream of the WAGO/22G-RNA system in transposon control. The specifics were rather hazy though. The 21U-RNAs matching Tc3 sequence were both orientated sense to the transposase gene, and so would not be able to base-pair with the transposase mRNA. The 21U-RNA identified by Batista et al. wasn’t even directed against a part of the transposon expected to be transcribed. However, the 22G-RNAs that were sensitive to prg-1 function were generally antisense to the direction of transposase transcription. These findings suggested a model in which transcription from downstream genomic regions may generate antisense Tc3 transcripts which would be recognised by a PRG-1/21U-RNA complex. This would then trigger the RdRP-dependent synthesis of 22G-RNAs against the transposon transcripts, that would then silence the transposons most probably through alterations to chromatin structure.

Linking the PRG-1/21U-RNA system to the WAGO/22G-RNA was a major step, and could be a general mechanism for 21U-RNA action. The 22G-RNA mediated stage of the process can be viewed as equivalent to the ping-pong amplified secondary piRNA stage in Drosophila transposon silencing. However, these findings raised important questions; If this is a system for the control of mobile elements, why was only one transposon found to be desilenced in prg-1 mutants, and so few 21U-RNAs found to match transposon sequences? And what about the vast number of 21U-RNAs without identity to other genomic sequences? A similar phenomenon is seen in mammals, in which a huge pool of ‘pachytene’ piRNAs without known targets or functions, are found. To explain this enigma, Batista et al. suggested that 21U-RNAs could base-pair imperfectly with their targets. microRNAs are able to recognise their targets by base-pairing within a ‘seed’ region driving less stringent pairing between the rest of the molecules. If 21U-RNAs work by a similar mode, the whole worm transcriptome could be under piRNA-directed regulation. Only modest changes in general gene expression were found in the prg-1 mutants, so it’s unclear what this global genome regulation or surveillance system really means.

I get the impression that a dichotomy of interpretations has arisen between the two principal labs studying 21U-RNAs. This can be broadly expressed as Eric Miska considering 21U-RNAs as a mechanism primarily directed against genomic parasites, whilst Craig Mello favours a broader ‘global surveillance system’ interpretation. The next post, on the recent papers from these labs, will try to dissect these different interpretations and clarify the current state of knowledge of C. elegans piRNAs.

Stop Press: A new paper has just cleared up the contentious issues of the function of the upstream motif, and whether 21U-RNAs are individually transcribed. Cecere et al. show that the upstream motifs are indeed promoters. Forkhead family transcription factors bind the motif, and drive the separate expression of the thousands of 21U-RNAs.

Ruby JG, Jan C, Player C, Axtell MJ, Lee W, Nusbaum C, Ge H, & Bartel DP (2006). Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell, 127 (6), 1193-207 PMID: 17174894

Das PP, Bagijn MP, Goldstein LD, Woolford JR, Lehrbach NJ, Sapetschnig A, Buhecha HR, Gilchrist MJ, Howe KL, Stark R, Matthews N, Berezikov E, Ketting RF, Tavaré S, & Miska EA (2008). Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline. Molecular cell, 31 (1), 79-90 PMID: 18571451

Batista PJ, Ruby JG, Claycomb JM, Chiang R, Fahlgren N, Kasschau KD, Chaves DA, Gu W, Vasale JJ, Duan S, Conte D Jr, Luo S, Schroth GP, Carrington JC, Bartel DP, & Mello CC (2008). PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Molecular cell, 31 (1), 67-78 PMID: 18571452

Cecere G, Zheng GX, Mansisidor AR, Klymko KE, & Grishok A (2012). Promoters Recognized by Forkhead Proteins Exist for Individual 21U-RNAs. Molecular cell PMID: 22819322

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)