RNA Journal Club 3/26/09
Elizabeth H. Bayne, Manuela Portoso, Alexander Kagansky, Isabelle C. Kos-Braun, Takeshi Urano, Karl Ekwall, Flavia Alves, Juri Rappsilber, Robin C. Allshire
Science 322 (5901): 602-606, October 2008.
This week’s paper dissection by David Weinberg:
Centromeres in fission yeast (Schizosaccharomyces pombe) consist of a central kinetochore domain flanked by heterochromatic outer repeats. These outer repeats are transcribed by RNA polymerase II and maintained in a heterochromatic state by the RNAi pathway. According to the current model of S. pombe RNAi, the RDRC complex (Rdp1, Hrr1, and Cid12) converts nascent outer repeat transcripts into dsRNA, which serves as a substrate for Dcr1. siRNA-loaded Ago1, in complex with Tas3 and Chp1, is directed to complementary nascent transcripts and stimulates H3K9-dimethylation of histones by the Clr4 histone methyltranferase.
A previous forward genetic screen for ts lethal defects in centromere silencing had identified two components of the spliceosome, Cwf10 and Prp39. Based on this evidence, the authors hypothesized that there may be a link between splicing and centromere silencing in S. pombe. In this paper, they take a reverse genetics approach to further investigate this potential connection.
The paper begins by characterizing the extent of centromere silencing in a collection of ts lethal splicing mutants. They find that only specific splicing mutants show a defect in centromere silencing, i.e. there are many splicing mutants that show no effect. Among their mutant collection, they find a strong correlation between silencing of a centromere-embedded reporter gene, transcript levels of endogenous outer repeats, and quantities of outer repeat-derived siRNAs.
At this point, the authors point out that there are many possible “mundane” explanations for the effects they see, namely that splicing itself may affect mRNAs encoding proteins that are directly/indirectly involved in RNAi (including potentially Ago1 itself). Their numerous attempts to disprove this potential artifact are commendable – thorough, clever, cautious. While their data alone cannot entirely rule out the mundane, they did as much as could be done to convince the reader (and reviewers, presumably) that there must be interesting (i.e. splicing-independent) science at work here.
Figure 3 contains, by far, the least novel/interesting experiments of the paper. Basically the authors demonstrate that the centromere silencing defect in splicing mutants is due to a minor disruption in the maintenance of RNAi-dependent heterochromatin. The effect they see is surprisingly weak in comparison to a dcr1-null strain, suggesting that the role of the spliceosome is not essential for RNAi-directed heterochromatin.
Luckily, the paper ends on a high note with Figure 4. They use mass spec to show that Cid12 interacts with the RDRC complex, and chromatin immunoprecipitation to demonstrate an association between the spliceosome and centromere repeat DNA. In this way, the authors convert a nebulous genetic interaction between splicing and RNAi into a physical interaction between the spliceosome and the RNAi machinery. This interaction is also consistent with Figure 4B, which suggest that splicing factors act downstream of RITS recruitment (e.g. at the level of RDRC-dependent siRNA amplification).
This final set of experiments hint at mechanism and lead the authors to speculate that the spliceosome may provide a platform that facilitates RDRC recruitment/action. While this model is completely consistent with the data, there is little direct support for this model over a variety of other consistent models (which they entirely ignore). Along these lines, I was most intrigued by their initial observation that only specific splicing mutants showed the centromere silencing defect…but there was not even a mention of how this relates to their model. Still, the novel aspects of this paper – namely, the link between the spliceosome, but not splicing, and RNAi in the form of a physical interaction between the spliceosome and RDRC – make this an important paper in the field. Given the absence of RdRP machinery in metazoans, it will be interesting to see if a similar splicing-RNAi interaction is at work in higher eukaryotes.