RNA Journal Club 10/29/09
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.
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.
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.