Tag Archives: siRNA

The CSR-1 siRNA pathway gets more enigmatic

A recent paper forces a reappraisal of the role of CSR-1 its associated 22G-RNAs, and demonstrates a positive regulatory role for this RNAi pathway in C. elegans.

As described in a previous post, depletion of the Argonaute protein CSR-1, or the proteins responsible for the biogenesis of the endo-siRNAs with which its complexes (the RdRP EGO-1, and the helicase DRH-3), results in defective mitotic chromosome segregation and sterility. To explain these findings Claycomb et al. proposed that the CSR-1 22G-RNA pathway acted to organise the proper compaction of the holocentric chromosomes of C. elegans, and the assembly of the kinetochores necessary for their proper segregation. (I strongly recommend reading the earlier post describing this paper’s findings).

Claycomb et al. had found that expression of most genes targeted by CSR-1 associated 22G-RNAs was not significantly altered in csr-1 mutants. Avgousti et al. went back over the same data and found that, although this was true in the main, expression of most of the genes encoding histone proteins was downregulated in csr-1 mutants. It had previously been shown that downregulation of just one histone gene could cause chromosome segregation and sterility phenotypes in worms. This lead Avgousti et al. to hypothesise that the defects seen in csr-1, ego-1 and drh-3 mutants may be caused by defective histone production, rather than the model proposed by Claycomb et al.

Histone proteins make up the core of the nucleosome and are multiply encoded in all eukaryotic genomes. Histone mRNAs are processed in a special way; generally their 3’UTRs are not polyadenylated; instead, downstream of a conserved stem-loop structure, a histone specific sequence (HDE) is recognised and cleaved by the U7 snRNA (an important splicing factor). Both HDE sequences and the U7 snRNA are not present in C. elegans. Avgousti et al therefore tested whether this key histone mRNA processing stage was instead being mediated by CSR-1 and its associated endo-siRNAs in worms.

Using a synthetic oligonucleotide identical to the region of the 3’UTR downstream of the stem-loop from the histone 2A pre-mRNA, they demonstrated that CSR-1 directly binds histone mRNAs. This binding was abrogated upon RNAi depletion of the RdRP EGO-1, showing that CSR-1 binding was dependent on the 22G-RNAs generated by EGO-1. Avgousti et al. also demonstrated that upon knockdown of CSR-1 or EGO-1, or in drh-3 mutants, unprocessed histone pre-mRNAs accumulate, whilst processed histone mRNAs and proteins are depleted.

The strongest evidence supporting the hypothesis that defective histone mRNA processing causes the defects seen in csr-1 mutants was a series of transgenic rescue experiments. Histone overexpression from transgenes, designed to not require 3’UTR mRNA cleavage, was able to counteract the effects of csr-1 or ego-1 RNAi knockdown, whereas transgenes that did required 3’UTR processing could not.

It seems likely therefore that in C. elegans the 3’UTR cleavage of histone pre-mRNAs is performed by CSR-1/22G-RNA complexes. CSR-1 has been shown to possess endonuclease ‘slicer’ activity, but although a likely candidate, it is too early to say whether it directly performs the cleavage or recruits other factors to perform the reaction. I think this paper blows a large hole in the model proposed by Claycomb et al. to explain the role of CSR-1 22G-RNAs; suggesting that the observed chromosome segregation defects are indirectly caused by a failure to produce adequate histones, rather than a failure to direct the organisation of mitotic chromosomes. However, the hypothesis certainly requires further and more subtle experiments. The paper also further muddies the waters on the question of just what the CSR-1 22G-RNA system is doing in most cases. The recognition of histone mRNA 3’UTRs can only account for a very small proportion of this endo-siRNA population. As discussed in other posts the CSR-1 22G-RNA system is the prime candidate to be an epigenetic licensing anti-silencing pathway. Do the two different CSR-1 isoforms perform two different functions; one licensing transcription and the other replacing the U7 snRNA splicing apparatus? Is this pre-mRNA splicing role confined to histone mRNAs? An important first of this paper is the demonstration of a positive role in regulating gene expression for an RNAi system. Generally, the various RNAi pathways negatively regulate gene expression; either resulting in slicing and degradation of transcripts, directing silencing chromatin modifications etc. In this case mRNA processing by the CSR-1 endo-siRNA system leads to proper expression of histones at key periods of rapid cell division (eg. early embryogenesis). Personally, I’m looking forward to more contentious interpretations of this pathway from the research groups involved!

Avgousti DC, Palani S, Sherman Y, & Grishok A (2012). CSR-1 RNAi pathway positively regulates histone expression in C. elegans. The EMBO journal PMID: 22863779

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 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)