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

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