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.

qiRNA is a new type of small interfering RNA induced by DNA damage

Heng-Chi Lee, Shwu-Shin Chang, Swati Choudhary, Antti P. Aalto, Mekhala Maiti, Dennis H. Bamford & Yi Liu

Nature 459: 274-277, 14 May 2009.
doi:10.1038/nature08041

This week’s aufschlussreiche summary and analysis by Anna Drinnenberg:

The term “quelling” refers to a posttranscriptional gene silencing phenomenon observed in Neurospora crassa, and was one of the first RNAi pathways to be described (Romano and Macino, 1992). Quelling is triggered by the expression of transgenes, also called “aberrant RNAs,” and results in silencing of both transgenes and cognate endogenous transcripts. It involves the production of double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (QDE-1) using the transgenic mRNA as a template. Subsequently, one of two Dicer proteins (DCL-1 and DCL-2) cleaves the dsRNA substrate into small RNA duplexes that get loaded into an Argonaute effector complex containing QDE-2. After cleavage and exonucleolytic digestion of the passenger strand, the other siRNA strand functions as a guide strand in QDE-2 to degrade homologous endogenous transcripts. The physiological role of quelling is thought to be control of transposon expansion in order to preserve genomic integrity.

The authors of this study suggest a new physiological role for components of the quelling pathway in the response to DNA damage. During the process of studying the regulation of QDE-2 they noticed that the expression of QDE-2, at both the mRNA and protein level, is upregulated upon DNA damage caused by adding Histidine, EMS, or Hydroxyurea to the media. Immunoprecipitating QDE-2, they identified a new class of small RNAs ~21nt in length whose abundance is increased in the IP following DNA damage. Interestingly, these small RNAs appear to be shorter than the previously identified siRNAs (~25nt) of the quelling pathway that are produced by the same Dicer proteins (Catalanotto et al., 2004). It will be interesting to determine if the Dicer proteins, that are thought to act redundantly (Catalanotto et al., 2004), can produce small RNAs of different lengths or if the interaction with a cofactor could determine the cleavage interval on the dsRNA substrate.

Most of the small RNAs, which they referred to as “qiRNAs,” are derived from the sense and antisense strands of an rDNA array exceeding the regions that are transcribed into rRNA by Pol1, suggesting that a distinct transcript gives rise to the precursor (aberrant RNA) for the qiRNAs. They noticed that the production of aberrant RNA was not inhibited by thioelutin, a known inhibitor of RNA polymerases. In an attempt to identify the protein that produces the initial qiRNA precursor transcript from the rDNA array, they observed that QDE-1, already known to have RdRP catalytic activity, can also synthesize RNA transcripts using a DNA template. This is a very interesting observation and raises the question if RdRPs in other organisms also have DNA-dependent RNA polymerase activity. Such an activity would make the production of precursor RNAs for small RNAs independent from the canonical transcription pathway for the majority of other cellular RNAs.

In trying to assign a role to the qiRNAs, the authors noticed that the decrease in protein production upon DNA damage is partially blocked in QDE-1 and QDE-3 mutant strains. Moreover, QDE-1 and DCL-1/DCL-2 mutants show increased sensitivity to DNA damage reagents. These observations certainly provide a first hint of function of this pathway. A more detailed follow-up experiment could be a more precise demonstration that the qiRNAs in complex with QDE-2 directly downregulate rRNA transcripts (but this experiment was beyond the scope of this study).

Another recent publication from Cerere and Cogoni (Cecere and Cogoni, 2009) suggests that the small RNAs are involved in copy number control of the rDNA locus, possibly preventing recombination within the array. Changes in the heterochromatic state of the rDNA array could unify both observations: the block in downregulation of the transcripts and the increased recombination rate. Moreover, high-throughput sequencing of the small RNAs as well as RNAseq analysis in Neurospora crassa upon DNA damage might also identify other qiRNA sources and their potential targets.

References:
Catalanotto, C. et al. (2004). Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24, 2536-2545.

Cecere, G., and Cogoni, C. (2009). Quelling targets the rDNA locus and functions in rDNA copy number control. BMC Microbiol 9, 44.

Romano, N., and Macino, G. (1992). Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6, 3343-3353.