You'd Prefer An Argonaute

Distinct Argonaute-Mediated 22G-RNA Pathways Direct Genome Surveillance in the C. elegans Germline

Weifeng Gu, Masaki Shirayama, Darryl Conte, Jessica Vasale, Pedro J. Batista, Julie M. Claycomb, James J. Moresco, Elaine M. Youngman, Jennifer Keys, Matthew J. Stoltz, Chun-Chieh G. Chen, Daniel A. Chaves, Shenghua Duan, Kristin D. Kasschau, Noah Fahlgren, John R. Yates, Shohei Mitani, James C. Carrington and Craig C. Mello

Molecular Cell 36: 231-244, 1 October 2009.
doi:10.1016/j.molcel.2009.09.020

This week’s comprehensive summary/analysis by Michael Nodine:

Upon screening for mutants defective in RNAi, Craig Mello’s group found that the DICER-RELATED HELICASE-3 (DRH-3) gene was required for germline and soma RNAi. Gu et al. also found that DRH-3 was required for endogenous siRNA (esiRNA) production and/or stability.  When examining the requirement of DRH-3 for esiRNA production more closely, they found that DRH-3 was involved in the production of a specific class of esiRNAs, which they termed the 22G-RNAs due to their length and preference for a 5’ guanosine. 22G-RNAs were found to not have 5’-monophosphates, which are typically found in DICER products, and this observation led the authors to hypothesize that 22G-RNAs are RNA-dependent RNA polymerase (RdRP) products rather than DICER products. They then cloned small RNAs from wild-type and drh-3 samples using a 5’-independent ligation method, and found that 22G-RNAs mapped to ~50% of the protein coding genes annotated in the C. elegans genome.

Interestingly, 22G-RNAs tended to map to the 3’-ends of genes and there was less of a requirement for DRH-3 for 22G-RNAs derived from gene 3’-ends. Since the drh-3 mutants contained point mutations in the conserved helicase domain, this hinted at the possibility that DRH-3 may be part of an RdRP complex and may facilitate its movement along the RNA template by removing inhibitory secondary structures. Consistent with this idea, they found that two RdRPs were redundantly required for 22G-RNA production, and that these two RdRPs along with the tudor domain-containing protein EKL-1 interacted with DRH-3.

They then went on to find that worm-specific Argonautes (WAGOs) were redundantly required for 22G-RNA production. Genes, transposons, pseudogenes and cryptic loci were all found to be targets of 22G-RNAs, and components of the non-mediated decay (NMD) pathway were demonstrated to play a role in the biogenesis of at least a subset of 22G-RNAs. Gu et al. also demonstrated when and where 22G-RNAs function during worm development. WAGO-1 was localized to P-granules, which are localized just outside nuclear pores in the female germline and are thought to play a role in maternal RNA repression and storage. In addition, high-throughput sequencing and developmental northerns suggested that 22G-RNAs are enriched in the female germline and maternally inherited.

Thus, 22G-RNAs are key components of a surveillance pathway, which operates in the female germline and represses protein coding genes, pseudogenes and transposons. Presumably, incorrectly processed protein coding transcripts are targets for 22G-RNA biogenesis/action. However, it remains unknown how aberrant transcripts are recognized. Transcripts lacking poly(A) tails were previously demonstrated to be better substrates for C. elegans RdRPs in vitro, and incorrectly processed transcripts are better substrates for RdRP-dependent RNAi in plants. Fission yeast nucleotidyl transferases have been implicated in the recognition of aberrant transcripts by RdRPs and exosomes, and a homologous nucleotidly transferase, as well as a 3’-5’ exonuclease were found to be required for 22G-RNA production.  Based on these observations, the authors suggest that a nucleotidyl transferase and a 3’-5’ exonuclease, both of which were shown to be required for 22G-RNA production, may function in an exosome-like complex to recognize aberrant transcripts and/or recruit the 22G-RNA RdRP complex. Finally, 22G-RNA pathway components are subcellularly positioned just outside female germline nuclei. Based on their observations, the authors hypothesize that 22G-RNA components may ‘monitor’ the female germline transcriptome and thus function in the surveillance of maternally-inherited RNAs.