The nucleolar transcriptome regulates Piwi shuttling between the nucleolus and the nucleoplasm
The nucleolus contains a lot of proteins unrelated to ribosome biogenesis. Some of these proteins shuttle between the nucleolus and the nucleoplasm regulating the cell cycle and stress response. The piRNA binding protein Piwi is involved in silencing of transposable elements (TEs) in the Drosophila gonads. Here we used cultured ovarian somatic cells (OSC) to characterize Piwi as a visitor to the nucleolus. Dynamic Piwi localization was shown to vary from its uniform distribution between the nucleoplasm and the nucleolus to pronounced nucleolar immobilization. We were intrigued by this localization behavior and revealed that nascent nucleolar transcripts recruit Piwi for nucleolar retention. Piwi eviction from the nucleolus was observed upon RNase treatment and after RNA polymerase (Pol) I inhibition, but not after Pol II inactivation. On the contrary, heat shock caused drastic Piwi redistribution from the nucleoplasm to the nucleolus, which occurred only in the presence of Pol I-mediated transcription. These results allow us to hypothesize that specific stress-induced transcripts made by Pol I promote the nucleolar sequestration of proteins in Drosophila, similar to previous observations in mammalian cells. We also found that in OSC, Piwi partially restricts expression of the rDNA copies containing R1 and R2 retrotransposon insertions especially upon heat shock-induced activation of these copies. Therefore, we suggest that Piwi intranuclear shuttling may have a functional role in ensuring a balance between silencing of rDNA-specific TEs under stress and the canonical Piwi function in non-nucleolar TE repression.
KeywordsPiwi Nucleolus rDNA Pol I Transposable elements Ovarian somatic cells
Ovarian somatic cells
We are grateful to Mikiko Siomi for providing OSC line, myc-Piwi vector and help in the implementation of this work; Artem Ilyin for assistance in performing Piwi knockdowns; Elena Fefelova for helpful advice on Piwi-GFP; Igor Kireev for assistance with EU incorporation assay; Konstantin Panov for providing 9-hydroxyellipticine inhibitor, and Michael Buszczak for Udd antibody. The work was carried out with the use of the equipment of the common use center “Center of Cell and Gene Technology”, Institute of Molecular Genetics, RAS.
Authors’ contribution statement
EM, VG, and MK conceived and designed the study; EM, TL, and HI performed research; EA, VG, and MK analyzed data; and VG and MK wrote the paper.
The work was supported by the Russian Foundation for Basic Research (grant no. 18-54-50015 YaPh_a) and by the Presidium of the Russian Academy of Sciences program Molecular and Cell Biology for VAG.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Barckmann B, Pierson S, Dufourt J, Papin C, Armenise C, Port F, Grentzinger T, Chambeyron S, Baronian G, Desvignes JP, Curk T, Simonelig M (2015) Aubergine iCLIP reveals piRNA-dependent decay of mRNAs involved in germ cell development in the early embryo. Cell Rep 12:1205–1216. https://doi.org/10.1016/j.celrep.2015.07.030 CrossRefGoogle Scholar
- Eickbush TH, Eickbush DG (2015) Integration, regulation, and long-term stability of R2 retrotransposons. Microbiol Spectrum 3: MDNA3–0011-2014 doi: https://doi.org/10.1128/microbiolspec.MDNA3-0011-2014
- García-López J, Alonso L, Cárdenas DB, Artaza-Alvarez H, Hourcade Jde D, Martínez S, Brieño-Enríquez MA, Del Mazo J (2015) Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization. RNA 21:946–962. https://doi.org/10.1261/rna.048215.114 CrossRefGoogle Scholar
- Ilyin AA, Ryazansky SS, Doronin SA, Olenkina OM, Mikhaleva EA, Yakushev EY, Abramov YA, Belyakin SN, Ivankin AV, Pindyurin AV, Gvozdev VA, Klenov MS, Shevelyov YY (2017) Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells. Nucleic Acids Res 45:7666–7680. https://doi.org/10.1093/nar/gkx355 CrossRefGoogle Scholar
- Iwasaki YW, Siomi MC, Siomi H (2015) PIWI-interacting RNA: its biogenesis and functions. Annu Rev Biochem 84:405–433. https://doi.org/10.1146/annurev-biochem-060614-034258 CrossRefGoogle Scholar
- Nollen EA, Salomons FA, Brunsting JF, van der Want JJ, Sibon OC, Kampinga HH (2001) Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection. Proc Natl Acad Sci U S A 98:12038–12043. https://doi.org/10.1073/pnas.201112398 CrossRefGoogle Scholar
- Osouda S, Nakamura Y, de Saint Phalle B, McConnell M, Horigome T, Sugiyama S, Fisher PA, Furukawa K (2005) Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation. Dev Biol 284:219–232. https://doi.org/10.1016/j.ydbio.2005.05.022 CrossRefGoogle Scholar
- Panse SL, Masson C, Heliot L, Chassery JM, Junera HR, Hernandez-Verdun D (1999) 3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription. J Cell Sci 112(Pt 13):2145–2154Google Scholar
- Pederson T (2011) The nucleolus. Cold Spring Harb Perspect Biol 3:a000638. https://doi.org/10.1101/cshperspect.a000638
- Wang M, Tao X, Jacob MD, Bennett CA, Ho JJD, Gonzalgo ML, Audas TE, Lee S (2018) Stress-induced low complexity RNA activates physiological amyloidogenesis. Cell Rep 24:1713–1721.e4. https://doi.org/10.1016/j.celrep.2018.07.040
- Welch WJ, Suhan JP (1985) Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol 101:1198–1211. https://doi.org/10.1083/jcb.101.4.1198 CrossRefGoogle Scholar