Chromosomal Flip-Flop

A study describes how phenotypic switching in Staphylococcus aureus is caused by a reversible large-scale genomic inversion.

Clonal bacterial populations often display various phenotypes. This diversity is most obviously identifiable as colony variation. Many different bacterial genera display ‘small colony variants’ (SCVs), the occurrence of which is generally attributable to deficiencies in various metabolic pathways.

Cui et al have characterised an STV strain of Staphylococcus aureus which reverts to a normal colony variant (NCV) at a frequency of 1-3 in a 1000. Interestingly the NCV progeny revert back to SCV in 1-10% of cases. This frequent bi-directional reversion was stably maintained at these ratios; homogeneous colony populations could never be isolated.

The small colony variant displayed some important phenotypic differences to the NCV. As well as slow growth and less pigmentation, it was susceptible to β-lactam antibiotics whilst the NCV was not. The authors identified over a hundred genes were differentially expressed between the two variants, and that their susceptibilities to many chemicals were different.

Diagram showing reversible genomic inversion forms caused by homologous recombination at inverted repeat regions (break points. BPs)

When Cui et al. sequenced the genomes of the two variants, they discovered that nearly half of the genome (1.26 Mb of 2.87 Mb) was differenttly aligned. This ‘X-shaped’ chromosome inversion occurred between two oppositely oriented pathogenicity islands, symmetrically opposite each other on the chromosome with respect to the replication axis. Each pathogenicity island contained two copies of an identical 3,638bp long sequence. It appears that homologous recombination can occur at these sites and generate the genomic inversion. This is in agreement with experiments in which the authors altered levels of the key recombination regulatory protein RecA; finding that they could increase the rate of reversion with higher recA expression.

The chromosomal flip-flopping therefore regulates the maintenance of two different S. aureus phenotypic variants. The two forms have different advantages and disadvantages. The original SCV strain isolated from a patient suffering persistent reinfection of a surgical site. It appears that the SCV may facilitate immune evasion, whilst the NCV has higher antibiotic resistance. Maintaining a balance between the two variants within the S. aureus population therefore functions as an evolutionarily useful bet-hedging strategy.

This type of flexible genome organisation serving as a self-organising regulatory mechanism for the maintenance of a bi-stable heterogeneous cell population may well be a more wide-spread bacterial evolutionary strategy.

Cui L, Neoh HM, Iwamoto A, & Hiramatsu K (2012). Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109 (25) PMID: 22645353

Expanding the Conjugative Realm

A recent paper demonstrates that a broader range of plasmids can be transferred by conjugation than previously thought.

Integrative and conjugative elements (ICEs, aka conjugative transposons) are a variety of bacterial mobile element generally found integrated into the host genome, but capable of excision and transfer to other cells via conjugation. I’ve previously written a short review of some of their key features, which may serve as a helpful introduction to this post. As well as transferring themselves between cells, ICEs and conjugative plasmids can mobilise other DNA elements, such as integrative mobilizable elements (IMEs) and mobilizable plasmids, that aren’t independently capable of self-transfer.

The conjugative transfer of any of these elements generally rests upon the generation of a single-stranded DNA molecule from the circular dsDNA mobile genetic element. The ssDNA is formed by a mobilising relaxase (Mob) nicking the circular DNA at an origin of transfer (oriT) sequence, followed by the unwinding of the strands by a host-derived helicase. Mob covalently binds the nicked end forming the ‘relaxosome’. A coupling protein is responsible for targeting the relaxosome to the conjugative apparatus (a type 4 secretion system, T4SS).

Plasmids that are incapable of self-transfer, but can be mobilised in trans by conjugative elements, generally encode their own mobilising relaxase and a cognate oriT site. These functions are separate from their replication system, which does however consist of similar components; a replication relaxase (Rep) which nicks and binds to an origin of replication (ori).

Lee et al. have discovered that three plasmids, which had been classified as non-mobilisable due to their lack of Mob/oriT functions, can in fact be transferred between Bacillus subtilis cells by the conjugation system of an ICE, ICEBs1. The three plasmids’ (pC194, pBS42, and pHP13) transfer required the conjugation machinery and coupling enzyme (ConQ) of ICEBs1, but was not dependent on it’s relaxase (NicK). Transfer could occur concomitantly with that of ICEBs1 or in it’s absence; showing that transfer did not act in cis due to integration of the plasmid into ICEBs1.

The authors found that, in the case of pBS42, it’s replicative relaxase was necessary for mobilisation. They therefore think it likely that in all three cases the Rep/ori system is also capable of mediating mobilisation functions. This blurring of the distinctions between Rep/ori and Mob/oriT systems has important ramifications. It opens up the possibility that many rolling-circle replicating plasmids that had been classed as non-mobilisable can in fact be transferred between cells via conjugation.

From an evolutionary perspective, these findings are important for understanding the persistence of plasmids in bacterial populations. Non-mobilisable plasmids would only be maintained in the population because benefits conferred on their hosts. If however many more ‘non-mobilisable’ plasmids can be disseminated by horizontal transfer, their persistence can be better explained. This study therefore expands the importance of conjugation in understanding bacterial evolution. Future studies will need to analyse the extent of interactions between coupling proteins and replication relaxases to better gauge the trans-mobilisation of genetic elements, and re-classify the mobility status of many plasmids.

Lee CA, Thomas J, & Grossman AD (2012). The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions. Journal of bacteriology, 194 (12), 3165-72 PMID: 22505685

Epigenetic Licensing of a Sex Determination Gene

A recent series of papers describing interacting small RNA pathways in C. elegans, posited the existence of an anti-silencing pathway responsible for licensing the expression of germline transcripts. Johnson and Spence (Science. 2011) published the first paper suggesting such a pathway. Using an elegant series of genetic experiments, they found that maternally supplied RNA of the sex determination gene fem-1 was necessary for normal zygotic fem-1 activity. In the absence of this maternal component, normal fem-1 alleles are epigenetically silenced.

In C. elegans, sex is determined by the ratio of sex chromosomes to autosomes. Worms with one X chromosome become males, whilst those with two develop as self-fertile hermaphrodites. Mutations in genes in the sex determination pathway can give rise to true females. For instance, fem-1 is required for masculinization in both males and hermaphrodites; worms deficient for both maternal and zygotic fem-1 activity lack all male development.

Johnson and Spence noticed some discrepancies between the relative importance of the maternal and zygotic components of fem-1 action. Homozygous fem-1 mutants, born of heterozygous mothers show partial male development because of maternally supplied fem-1. Zygotically heterozygous progeny of female worms homozygous for point mutations in fem-1 are phenotypically normal; that is to say that despite the lack of any maternally derived active FEM-1 protein, male development can proceed normally. However, similar crosses, in which fem-1 deletion alleles are used rather than point mutations, give rise to heterozygous worms showing feminization of the germ line (Fog phenotypes). To prove the requirement of a maternal fem-1 product for spermatogenesis, Johnson and Spence performed a clever genetic trick that produced worms that were homozygous for the wild-type fem-1 allele but were born of a mother homozygous for the deletion mutation. Most of these worms also exhibited Fog phenotypes. Other strong loss of function alleles that eliminated FEM-1 protein, could complement (ie. rescue) the deletion mutation-caused maternal effect. It therefore appeared that maternally derived fem-1 RNA was required for spermatogenesis, independently of its protein-coding function.

To confirm this, the authors injected in vitro transcribed fem-1 RNA into females homozygous for the fem-1 deletion allele. These worm’s progeny displayed fewer Fog phenotypes than those of uninjected worms. A similar level of rescue was observed when fem-1 RNA without a translation initiation codon was injected, showing that FEM-1 protein was not required.

What was the function of the maternal fem-1 RNA? Johnson and Spence hypothesised that it might be required for normal zygotic fem-1 activity. They therefore assayed this in heterozygotic worms descended either from mothers homozygous for the deletion, or from heterozygotes. Both a genetic test and observation of zygotic fem-1 RNA levels showed that zygotic fem-1 activity in the germline was impaired in the absence of maternal fem-1 RNA.

The authors went on to show that the severity of the Fog phenotypes observed in heterozygotes depended on the history of the wild-type fem-1 allele. Repeatedly backcrossing heterozygotes with homozygotes increased the penetrance of Fog phenotypes in heterozygotes. This showed that although the wild-type allele remains the same genetically, it becomes heritably compromised epigenetically.

To explain these findings, the authors suggest that in the absence of maternal fem-1 RNA, the paternally contributed wild-type allele becomes epigenetically silenced in the zygotic germline. They perspicaciously reasoned that the anti-silencing activity of the maternal RNA may work by inactivating silencing siRNAs, hence licensing zygotic expression. The cluster of recent papers (discussed here) have come to similar conclusions, suggesting the presence of an anti-silencing pathway, possibly mediated by CSR-1 and it’s associated 22G-RNAs.

This epigenetic licensing system effectively marks genes previously expressed in the germline as ‘self’, whilst transcripts without a history of expression are silenced as potentially deleterious. So far the mechanisms of this pathway are yet to be elucidated. As the CSR-1/22G-RNA system targets thousands of genes, one would expect similar phenomena to those observed with fem-1 to be more regularly observed. I’m looking forward to further findings in this field, and potentially analogous systems being discovered in other organisms.

Johnson CL, & Spence AM (2011). Epigenetic licensing of germline gene expression by maternal RNA in C. elegans. Science (New York, N.Y.), 333 (6047), 1311-4 PMID: 21885785

Trans-Extremophile Para-Sex

Cells of Haloferax volcanii showing their cup-like morphology

More than twenty years ago, an extra-ordinary mode of para-sexual genetic exchange was found to occur between cells of the halophilic archaeon Haloferax volcanii, an extremophile isolated from the Dead Sea. This process involves first, cell fusion producing heterodiploid cells. In this state, parental chromosomes can recombine, hence producing novel hybrid daughter cells after cell separation.

Haloferax mediterranei

Naor et al. have now shown that this mechanism of genetic exchange can also occur between cells of H. volcanii, and those of a related species – Haloferax mediterranei. H. mediterranei was originally isolated from a saltern near Alicante in Spain, and shares on average 86.6% sequence identity in protein-coding genes with H. volcanii. Assaying for chromosomal markers, it was found that interspecies cell fusion occurs at a frequency of 4.2 x 10^-5, less than one order of magnitude less frequently than between H. volcanii cells (1.0 x 10^-4). Of intraspecies fusions, 62% resulted in genetic recombination, whereas only 8% of interspecies fusions did. Although these figures sound quite small, in comparison to modes of genetic exchange between bacterial species, this inter-species genetic interchange occurred  a few orders of magnitude more efficiently.

Analysing a number of hybrid genomes, Naor et al. found that recombinant fragments ranged in size between 310 kb and 530 kb – huge genomic portions, with respect to most known bacterial horizontal genetic transfer mechanisms. Recombination occurs at areas higher sequence identity, such as transfer RNA genes. As these hybrids were isolated by selection for a specific genetic marker, it is quite conceivable, that higher proportions of the genomes can be transferred by this process.

This form of genetic exchange has important implications for archaeal speciation and evolution. High rates of recombination can act as a homogenising force – unlinking alleles faster than genes can diversify. The authors therefore suggest that geographical isolation may be the primary force in archaeal speciation. Further experiments testing the efficiency of this parasexual genetic exchange mechanism between other Haloferax species, and more information on halophilic archaeal ecology will help to clarify these issues. It will also be interesting to examine more hybrid genomes derived from other selection experiments to find out just how extensive interspecies recombination can be and whether there are any directional biases in transfer.

Naor A, Lapierre P, Mevarech M, Papke RT, & Gophna U (2012). Low species barriers in halophilic archaea and the formation of recombinant hybrids. Current biology : CB, 22 (15), 1444-8 PMID: 22748314

Genomic Rearrangement in Lampreys 2

As discussed in a recent post, during lamprey embryogenesis programmed genomic rearrangements lead to deletion of ~20% of the germline genome in the soma. Smith, Amemiya and co-workers have now published a follow-up study in which they further characterise the complement of deleted genes. Their findings have led them to hypothesise that the programmed genomic rearrangements (PGRs) serve to segregate pluripotency functions required in germline that could be deleterious in the soma.

Smith et al used a couple of different genomic techniques to identify somatically deleted sequences. Using microarrays constructed from available germline sequence, they found that ~13 % of the sampled sequence was deleted in the soma (in relative agreement with the ~20% derived from flow cytometry). Within this dataset, they identified 8 new single-copy/low-copy number sequences found only in the germline. RT-PCR showed that 5 of the novel sequences were expressed in germline cells. In situ hybridisation of one of these sequences showed that it was expressed in differentiating primordial germ cells in lamprey embryos.

The main limitation for identifying more genes subject to somatic deletion has been a lack of germline genomic sequence. Smith et al. performed high-throughput shotgun sequencing on lamprey sperm cells, generating short sequence reads covering ~10% of the germline genome. They then compared this dataset with the whole-genome sequence derived from somatic (liver) cells, yielding tens of thousands of putative deletion and recombination sites. A substantial part of the somatically deleted DNA corresponds to single-copy, protein-coding genes; the authors identified 246 instances of homology to individual human genes.

The problem with this comparison however, is that, by necessity, it was generated from 2 different individuals (of different sexes). This meant that apparent cases of deletion or recombination may be due to polymorphisms for insertion or deletion mutations present in lamprey populations. The researchers undertook validation experiments on a subset of the candidate deletion/recombination dataset (using PCR to amplify candidate sequences from testes and blood from 4 different males, blood from 4 different females, as well within an array of somatic tissue types within individuals). Of 48 tested candidate gene deletions or recombination events, they validated 7 sites of programmed deletion, and 3 recombination sites. They also identified 3 insertion/deletion polymorphisms, and 5 gaps in the somatic whole genome sequence. Due to PCR failures, or because of repetitive target sequences, 30 of the candidates were not informative. The validated gene deletions included APOBEC-1 complementation factor, encoding a protein involved in RNA editing, and the secreted developmental signalling molecule encoding WNT7A/B.

In the process of these validation experiments, Smith et al discovered short palindromic sequences at the deletion breakpoints. There was no specific consensus sequence at these positions, but the palindromes may indicate that the mechanism of chromatin diminution utilises site-specific recombination.

Another interesting finding of these experiments is that it appears that the programmed deletions are inherited uniformly throughout all the various somatic lineages. The earlier paper (discussed in the previous post) had suggested that different somatic tissues might have subtly different deleted portions. Microarray experiments, and comparison of the validated gene deletions between different tissues found no evidence of this, although this question may as yet not be answered definitively.

The crux of the paper rests on a computational comparison of ontology terms (in which homology is used to make predictions of cellular function, which are further sorted into broad categories). In the dataset of predicted gene deletions, certain ontologies were overrepresented with respect to the rest of the germline sequence; these included ‘regulation of gene expression’, ‘chromatin organisation’, and ‘development of germ/stem cells’.

Simply put, the paper has shown that a substantial number of protein-coding genes as well as repetitive sequences are deleted from the genomes of lamprey somatic cells. Many of the deleted genes are expressed in the germline, and often appear to have important regulatory functions. The crucial characteristics of the germline are the ability to undergo meiotic recombination, and totipotency. The missexpression of factors involved in these processes in the soma would be seriously detrimental; potentially resulting in aberrant cell fate specification, genome disruption, and hence cancers. The authors postulate that this conflict of interests between the germline and the soma underlies their genomic differentiation.

I find this an attractive and interesting hypothesis. As yet though, I don’t think the data is strong enough to have proved it. Gene ontology terms are relatively crude categorisations, and compounded with the question of what proportion of candidate deletions are bona fide, I’ll withhold judgement on the evolutionary rationale behind the deletions for the moment. Jeramiah Smith and colleagues are currently assembling the entire lamprey germline genome. Complete annotation of the deleted portion of the genome will certainly reveal the function of these fascinating genome rearrangements more clearly. I look forward to new studies investigating the mechanisms underlying the rearrangements, and their developmental progression. The extensive genome remodelling that occurs in ciliates utilises a combination of a small RNA/Argonaute system and domesticated transposase enzymes. I guess that analysis of any transposases encoded in the lamprey genome may be the place to start to unravel the mechanisms of chromatin diminution.

Smith JJ, Baker C, Eichler EE, & Amemiya CT (2012). Genetic consequences of programmed genome rearrangement. Current biology : CB, 22 (16), 1524-9 PMID: 22818913

Upstream ORFs and Regulation of Translation

Protein expression can be rapidly and responsively regulated at the level of translation. Translational regulation commonly involves trans-acting factors such as miRNA complexes or RNA-binding proteins, specifically binding cis-regulatory sequences. A study from last year – Medenbach et al. – has demonstrated an interesting mechanism in which translation of a transcript is repressed by the initiation of translation at a short upstream open reading frame (uORF).

Sexual organisms need a mechanism to balance the levels of transcription from sex chromosomes between sexes. In mammals this ‘dosage compensation’ is achieved by inactivating one of the X chromosomes in females; Drosophila instead hypertranscribes the single X chromosomes of male flies. In females, hypertranscription is prevented by the translational silencing of male-specific lethal (msl)-2 mRNA by a key sex determination factor, the RNA-binding protein Sex-lethal (SXL).

SXL exerts translational control on msl-2 transcripts by two distinct mechanisms. Binding in the 3’UTR, in conjunction with a co-repressor, it blocks the recruitment of the ribosomal pre-initiation complex to the 5’UTR. Any pre-initiation complexes that escape this control are then challenged by a failsafe mechanism; SXL bound to the 5’UTR causes destabilisation of the small ribosomal subunit upstream of the SXL-binding site.

Medenbach et al. analysed the mechanisms of this 5’UTR SXL-mediated translational control, using constructs in which altered msl-2 5’UTRs drove expression of a reporter gene in a cell-free system. The msl-2 5’UTR contains three small upstream ORFs. Mutations preventing initiation in the first two of these uORFs did not impair translational control, but when the third uAUG was mutated the reporter construct was translationally de-repressed. This effect was dependent on a SXL-binding site slightly downstream of the uORF, mutation of which abrogated the derepression. The repression was dependent on the presence of SXL. By itself the uORF reduced translation of the downstream reading frame two fold, but in the presence of SXL, downstream translation was repressed more than 14 fold.

The uORF encodes a di-peptide (Methionine-Threonine). By swapping the threonine-encoding codon and the subsequent stop codon, and by deleting the stop codon, the researchers showed that translational repression required uORF initiation and not translational elongation or termination.

Medenbach et al. went on to investigate just how widespread this mechanism of translational control may be. Computationally scanning the Drosophila transcriptome, they found that 58% of 5’UTRs contain one or more upstream initiation codons, whilst 4.3% contain putative SXL binding sites. 268 mRNAs (1.3%) contain both a uORF and a SXL-binding motif at the appropriate distance from one another to make them candidates for a similar mode of SXL-mediated translational repression. They then tested 12 of these candidate 5’UTRs in their reporter assay, and found that 6 of them did indeed mediate SXL-dependent translational repression. They also demonstrated that the regulatory cassette consisting of the uORF, the intervening 21nt, and the SXL-binding site was capable of mediating repression when inserted into the 5’UTR of an unrelated gene.

uORFs have previously been shown to regulate translation of the main reading fame in other contexts. The authors reference two examples using different mechanisms; a system involving termination and reinitiation from 4uORFs in a budding yeast transcript that doesn’t require any trans-acing factors; and another fungal mechanism in which ribosomal stalling at the termination codon of an uORF causes the mRNA to be degraded. In contrast, the translation regulation system in the msl-2 5’UTR utilises a binary module requiring only translational initiation at the uORF and binding of SXL. How exactly this works with respect to the ribosomal pre-initiation complex is not yet clear. Recognition of an initiation codon triggers a conformational change in this complex from a scanning ‘open’ structure to a closed conformation, followed by subunit joining to make a complete translationally competent ribosome. The authors found that SXL interacts with two subunits of the elF3 initiation factor, but weren’t able to prove the importance of these interactions for translational repression.

Medenbach et al’s computational scan of the Drosophila genome showed that the use of this binary module is an important mechanism by which SXL regulates the expression of many proteins. Similar uORF modules may be utilised by other RNA binding proteins, but it’s also possible that other uORFs function in quite different ways, as suggested by the examples from fungi. What’s beyond doubt, seeing as more than half of mammalian and Drosophila genes contain them is that uORFs are an important factor in translational regulation.

Medenbach J, Seiler M, & Hentze MW (2011). Translational control via protein-regulated upstream open reading frames. Cell, 145 (6), 902-13 PMID: 21663794

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 4^th 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 3: CSR-1 associated 22G-RNAs

Anaphase during the first mitosis of a C. elegans embryo. Microtubules (green) are pulling the two sets of daughter chromosomes (blue) towards the centrosomes (yellow).

During mitosis, duplicated chromosomes are separated and segregated into two daughter cells. This is achieved by the action of spindle microtubules. In metaphase, microtubules radiating from centrosomes at two poles in the cell, attach to the condensed chromosomes at proteinaceous structures called kinetochores. The daughter chromosomes are then pulled slowly towards opposite poles during anaphase. Mostly, eukaryotes have monocentric chromosomes – meaning that each chromosome contains one centromere. Kinetochores are associated with domains of heterochromatin close to the centromeres. This pericentric heterochromatin is composed of repetitive sequences, and is generally transcriptionally silenced.

An important feature of the mechanisms stabilising the heterochromatic state in some organisms are small RNAs. In the fission yeast Schizosaccharomyces pombe an RdRP-dependent population of small RNAs, directed against repetitive sequence, has been shown to target pericentromeric heterochromatin, stabilising the centromeres. Perturbations to this pathway therefore lead to mitotic chromosome segregation defects.

Not all organisms’ chromosomes contain single centromeres. Nematodes have holocentric chromosomes in which multiple spindle attachments are made into continuous kinetochores spanning the length of the chromosomes. The assembly of centromeres and kinetochores in both monocentric and holocentric chromosomes have many conserved features. For instance, the histone variant HCP-3/CENP-A is found in centromeric nucleosomes. Unlike in monocentric chromosomes though, in nematodes it is incorporated into the whole poleward face of condensed chromosomes.

In C. elegans, a number of mutations affecting components of RNAi pathways were found to cause mitotic and meiotic chromosome segregation defects. As discussed in the previous post, these included drh-3 (encoding a helicase necessary for the biosynthesis of 22G-RNAs), and the Argonaute protein encoding csr-1. Claycomb et al. found that mutations in two additional genes, the RdRP encoding ego-1, and the tudor domain protein encoding ekl-1, caused similar defects.  These included oocytes with abnormal complements of chromosomes, underproliferated germlines, and high incidence of males (him) phenotypes. Partial mutant alleles of csr-1 or drh-3 had low proportions of viable progeny, with many worms dying at various points in embryogenesis.

In embryos depleted of any of these factors, chromosomes initially condense normally in prophase. However, during metaphase the chromosomes fail to align into ‘plates’ perpendicular to the long axis of the spindles, and at anaphase the researchers observed chromosomal bridging across the midzone of the spindle. This led to the accumulation of cells with abnormal chromosomal complements, and the death of embryos.

Claycomb et al. then looked at the distribution of the centromeric histone variant HCP-3/CENP-A in RNAi depleted embryos. They found that although it was loaded onto chromosomes, instead of the normal poleward localisation, it was distributed in a disorganised pattern over the metaphase chromosomes. Similarly disorganised patterns were seen with an array of centromere and kinetochore proteins, as well as proteins necessary for chromosomal condensation and cohesion.

Deep sequencing of the small RNAs associated with CSR-1, showed that this argonaute binds 22G-RNAs targeted against protein-coding genes. This population of 22G-RNAs are dependent upon drh-3, ego-1, and ekl-1 and were antisense to at least 4191 genes. Interestingly, in glp-4 mutants that lack a germline, 22G-RNAs corresponding to ~80% of the CSR-1 target mRNAs were strongly depleted. This suggests that the CSR-1 associated 22G-RNAs originate in the germline and target genes expressed in the germline. No differences were found in the transcriptional profiles of csr-1 mutants and wild type worms, showing that CSR-1 22G-RNA system does not work by silencing its’ targets.

During the development of the germline, DRH-3, EGO-1 and CSR-1 all localise to P-granules – perinuclear nuage structures important for germline specification and small RNA mediated activities. In later stages of oocyte maturation CSR-1 becomes enriched in nuclei. In mitotic cells all four factors were enriched in prophase nuclei, and could be observed to localise to metaphase chromosomes. Using chromatin immunoprecipitation (ChIP), Claycomb et al. demonstrated that CSR-1 directly bound genomic loci targeted by CSR-1 associated 22G-RNAs.

These slightly disparate lines of evidence can be condensed into a basic model by which the CSR-1 22G-RNA pathway contributes to the regulation of chromosome segregation. CSR-1 associated 22G-RNAs are generated in the germline. Their biogenesis occurs in P-granules and depends on the RdRP complex (comprised of the helicase DRH-3, the RdRP EGO-1, and the tudor domain protein EKL-1) acting upon transcripts of germline expressed genes. Guided by these 22G-RNAs, CSR-1 translocates to the nucleus, and via chromatin modification defines chromosomal domains. It appears that there is an inverse relationship between domains targeted by CSR-1 22G-RNAs and those enriched for the centromeric histone variant HCP-3/CENP-A. Hence, CSR-1 marks domains adjacent to centromeric domains. As CSR-1 22G-RNA targets are distributed relatively uniformly across the genome, the defined domains can serve to position kinetochores along the length of the chromosomes. CSR-1 22G-RNAs are maternally inherited, and the CSR-1 defined chromatin domains are epigenetically stable, enabling the correct assembly of kinetochores during mitoses throughout development.

It is interesting to note that two related 22G-RNA pathways, those associated with WAGO or CSR-1 argonautes, with a similar biogenesis pathway, can have such diverse cellular roles. As we have seen the WAGO 22G-RNA pathway acts to silence deleterious transcription. The CSR-1 22G-RNA pathway doesn’t silence its’ targets but instead intricately organises chromosome structure to ensure proper segregation. Does this pathway have roles beyond this function? In later posts in this series I’ll cover papers that suggest it may also participate in a global genome surveillance network.

See also a post on a paper that gives a radically different take on the CSR-1 pathway: The CSR-1 siRNA pathway gets more enigmatic

Important ideas regarding the role of the CSR-1 pathway being involved in epigenetic licensing are discussed in these two posts:
Interacting small RNA pathways in worms 5: Global Genome Surveillance
Epigenetic Licensing of a Sex Determination Gene

Claycomb JM, Batista PJ, Pang KM, Gu W, Vasale JJ, van Wolfswinkel JC, Chaves DA, Shirayama M, Mitani S, Ketting RF, Conte D Jr, & Mello CC (2009). The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell, 139 (1), 123-34 PMID: 19804758

Interacting small RNA pathways in worms 2: 22G-RNAs

In two papers published in 2009 (Gu et al. and Claycomb et al), Craig Mello’s group characterised 22G-RNAs. They found that they could be divided into two different functional classes based on the Argonaute proteins with which they are complexed – CSR-1 or WAGOs. The biogenesis of both groups utilises common factors, but each targets different classes of loci and fulfil different roles.

The starting point for the discovery of 22G-RNAs was the analysis of various mutant alleles of the gene drh-3 (which encodes a dicer-related helicase). Homozygous drh-3 alleles result in infertile worms, whilst RNAi targeting leads to worms dying in embryogenesis with defects in chromosome segregation and in the production of small RNA populations. Gu et al identified three partial loss of function drh-3 alleles in which the homozygous worms were viable at 20°C but infertile at 25°C. When analysing the small RNA populations present in these worms, they found that a prominent 22nt species of RNA was absent, whilst others were unaffected. Chemical analysis showed that these RNAs had 5’ guanosine residues, and were 5’ triphosphorylated (most small RNAs are 5’ monophosphorylated – as produced by the activity of the Dicer endonuclease). The 22nt RNAs were not sensitive to periodate – suggesting that, unlike piRNAs they are not 3’ modified.

Gu et al. then made libraries of all small RNAs from wild type and drh-3 mutant worms, and deep sequenced them. 21nt and 22nt RNAs accounted for 25% and 36% of the wild type reads, respectively. The 21nt RNAs were divided equally between those with 5’U and those with 5’G. ~ 60% of the 22nt reads had a 5’G. Both 21nt and 22nt 5’G containing RNAs were strongly depleted in the drh-3 mutants. Of all the endogenous siRNA reads in the wild type library (64% of the total), ~53% were antisense to protein-coding genes, whilst ~16% were derived from transposons and repetitive sequences and ~31% were from non-annotated loci. All of these endo-siRNAs were depleted in drh-3 mutants. To try and clarify matters, Gu et al. termed this population of 22nt drh-3-dependent endo-siRNAs with a bias towards 5’G, 22G-RNAs. (note: I’m not quite clear as to whether this included 21G and/or 22U populations as well).

By analysing various mutant lines, Gu et al. found that 22G-RNAs are present in the soma and the germline. However, they were especially enriched in the germline, and in oocytes (ie. maternally derived). In the soma 22G-RNAs appear to act downstream of the exogenous-RNAi pathway (which won’t be discussed further – I’ll concentrate on their roles in the germline).

Germline 22G-RNAs are independent of the exo-RNAi pathway. For instance, they were not depleted in dcr-1 (dicer) mutants, suggesting that their biogenesis is not triggered by dsRNA. The triphosphorylated 5′ end of 22G-RNAs, and the independence from dcr-1, suggested that their biosynthesis was dependent on RNA-dependent RNA polymerases (RdRPs).  Single mutants for the known RdRPs still expressed 22G-RNAs. However, in worms mutant for both rrf-1 and ego-1, the researchers found that they failed to accumulate. Immunoprecipitation experiments showed that DRH-3 interacted biochemically with both RRF-1 and EGO-1, as well as a tudor-domain containing protein, EKL-1. These four proteins make up the core RdRP complex responsible for the biosynthesis of 22G-RNAs in the germline.

Depletion of Argonaute proteins leads to a reduction of the small RNAs with which they complex. Gu et al. used worm lines mutant for multiple WAGO genes to get a picture of which AGOs mediated 22G-RNA function. They found that worms deficient in wago-1 showed a major reduction in germline 22G-RNAs, whilst a worm strain lacking all 12 wago genes (MAGO12) showed an even greater deficit.

Deep sequencing from worms mutant for drh-3, ekl-1, or from the rrf-1 ego-1 double mutants had near complete germline 22G-RNA deficits. However in the MAGO12 worms, only a subset of 22G-RNAs matching repeat elements, as well as some coding and non-annotated loci were absent. Gene-targeted 22G-RNAs were far less likely to be affected. Immunoprecipitation of WAGO-1 complexes revealed an enrichment for the repeat element biased subset, whilst the AGO CSR-1 was found to interact with a subset of  22G-RNAs that are antisense to germline-expressed protein coding genes (Claycomb et al. discussed next post). This bimodal distribution of 22G-RNA targets revealed that two distinct 22G-RNA pathways functioned in the germline. They both share a common biosynthesis pathway but differ in the AGOs with which they complex.

The WAGO-associated 22G-RNA pathway appears to act by silencing it’s targets. Those loci targeted by the most highly expressed 22G-RNAs were derepressed in drh-3 mutants. Transposons are a major target for the WAGO mediated system. 22G-RNAs matching repetitive elements were depleted in MAGO12 worms. By assaying the reversion rate of mutations caused by the insertion of the transposon Tc5, and by monitoring the transcription of the Tc1 and Tc3 transposons, Gu et al showed that transposons are derepressed in drh-3 mutants (I’d have preferred to see these effects in the MAGO12 mutants to definitively show that it’s only the WAGO associated subset required).

The WAGOs are a worm specific clade of AGOs which don’t seem to act by ‘Slicer’ endonuclease activity. This study showed a lot of redundancy amongst WAGOs with regard to their 22G-RNA associated roles. However, the authors expect there to be a number of distinct roles within this family of factors. WAGO-1 appears to be a crucial factor in these systems. Importantly, this paper showed the existence of a major Dicer-independent RNA based genome surveillance system. This system has the ability to silence transposons and other repetitive sequences. It also appears to act upon pseudogenes and ‘cryptic loci’, preventing detrimental transcription/translation. However, the details of this system’s targets were beyond of the scope of this first paper.

The next post will discuss CSR-1 associated 22G-RNAs, before we come to 21U-RNAs and the links between the three systems.

Gu W, Shirayama M, Conte D Jr, Vasale J, Batista PJ, Claycomb JM, Moresco JJ, Youngman EM, Keys J, Stoltz MJ, Chen CC, Chaves DA, Duan S, Kasschau KD, Fahlgren N, Yates JR 3rd, Mitani S, Carrington JC, & Mello CC (2009). Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Molecular cell, 36 (2), 231-44 PMID: 19800275

Claycomb JM, Batista PJ, Pang KM, Gu W, Vasale JJ, van Wolfswinkel JC, Chaves DA, Shirayama M, Mitani S, Ketting RF, Conte D Jr, & Mello CC (2009). The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell, 139 (1), 123-34 PMID: 19804758