Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin

Schubert, Thomas; Pusch, Miriam Caroline; Diermeier, Sarah D.; Beneš, Vladimír; Kremmer, Elisabeth; Imhof, Axel; Längst, Gernot

doi:10.1016/j.molcel.2012.08.021

Cited by 115 publications

(109 citation statements)

References 34 publications

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“…In recent years, snoRNAs have been implicated in a wide range of pathways, suggesting that they have functions beyond their roles in RNA modification. snoRNAs maintain chromatin accessibility (36), regulate the coactivator activity of the dyskerin RNP (37), interact with Dicer (38), and are associated with disease (39). Using multiple assays, we show that snoRNAs interact with PKR in a PA-dependent manner.…”

Section: Discussionmentioning

confidence: 97%

Potential role for snoRNAs in PKR activation during metabolic stress

Youssef

Safran

Nakamura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

104

103

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Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR WT ) or PKR with a point mutation in each dsRNA-binding motif (PKR RM ). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR WT samples after PA treatment, but not in the PKR RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.PKR | snoRNA | metabolic stress | phosphorylation | RNA-binding protein

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Section: Discussionmentioning

confidence: 97%

Potential role for snoRNAs in PKR activation during metabolic stress

Youssef

Safran

Nakamura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

104

103

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“…2.5 μg of total RNA was heat fragmented for 9 min at 90ºC. The binding assay was performed as previously described (25). In brief, increasing amounts of recombinant His-KDM4D-FL was incubated with the fragmented RNA in RNA binding buffer (20 mM Tris-HCl, pH 7.6, 1.5 mM MgCl 2 , 80 mM KCl, 0.5 mM EGTA, 10% glycerol) for 30 min at RT.…”

Section: Methodsmentioning

confidence: 99%

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

Zoabi

Nadar‐Ponniah

Khoury-Haddad

et al. 2014

Nucleic Acids Research

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The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.

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“…Either the intrinsic fluorescence of tryptophanes within proteins [27] or the fluorescence signal of a fluorophore [28,29], attached to one of the interaction partners is monitored. Fig.…”

Section: Microscale Thermophoresismentioning

confidence: 99%