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In Pneuli et al 2015, they knock down an enhancer RNA using RNAi, testing whether it is a mere byproduct or whether it has a key role in the enhancer's function. They find their system works: the siRNA knocks down the enhancer RNA that it's targeted to, enabling further study. Hooray!

... but, I've read that RNAi and the RISC work outside the nucleus, and intra-nuclear effects are further downstream and indirect (example: Gosline et al. assume that effects on introns are indirect). I assume the paper's not wrong, so what's going on?

  • Conventional wisdom is wrong or incomplete: RNAi can work inside the nucleus too.
  • Pneuli et al's enhancer RNA gets exported to the cytoplasm and then re-imported to act on its target gene.
  • Something else?

Pnueli, L., Rudnizky, S., Yosefzon, Y., & Melamed, P. (2015). RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin α-subunit gene. Proceedings of the National Academy of Sciences, 112(14), 4369-4374.

Gosline, S. J., Gurtan, A. M., JnBaptiste, C. K., Bosson, A., Milani, P., Dalin, S., ... & Fraenkel, E. (2016). Elucidating microRNA regulatory networks using transcriptional, post-transcriptional, and histone modification measurements. Cell reports, 14(2), 310-319.

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    $\begingroup$ Are you sure an antisense RNA against this enhancer RNA uses the cell’s microRNA RISC system? I don’t know, but a priori would expect not. $\endgroup$
    – David
    Sep 5 '21 at 18:09
  • $\begingroup$ I have no idea. What else would it use? $\endgroup$ Sep 5 '21 at 18:54
  • $\begingroup$ Hybridization? When antisense RNA was first introduced the microRNA Dicer system was unknown, and the strategy was based on the idea of inactivating mRNAs by hybridization. The reason I would not expect such antisense RNAs to use the Dicer system is that they do not have the imperfect stem-loop structure of the natural substrate. I may be wrong as this is not a technique I have used, but you should check it out before assuming your assumption is correct and there is a problem with the paper $\endgroup$
    – David
    Sep 5 '21 at 19:28
  • $\begingroup$ Would hybridization lower concentration of the target RNA as measured by e.g. qPCR? Or would it affect activity without altering concentration? $\endgroup$ Sep 5 '21 at 19:46
  • $\begingroup$ FWIW, the system they describe using is shRNA, which I thought worked via RNAi. That's what wikipedia says anyway. If it's more complicated than that, well, that's basically my question. $\endgroup$ Sep 5 '21 at 19:47
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RNAi can happen in the nucleus as well. This is better documented in C.elegans but there are some references supporting nuclear RNAi in mammalian cells too:

  1. https://doi.org/10.1073/pnas.1707440114
  2. https://doi.org/10.1016/j.celrep.2013.12.013
  3. https://doi.org/10.1093/nar/gkv1305
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