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Convergence of distinct RNA-silencing pathways on GW182/Tnrc6

ABSTRACT: The RNA-binding protein Trim71/Lin41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development and cancer. Trim71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Deposited data are mass spectrometry data from immunoprecipitation experiments with endogenously tagged Trim71, Ago2, and Tnrc6a. Experiments are carried out in mouse embryonic stem cells (mESCs) and uncover reciprocal interactions of Trim71, Ago2, and Tnrc6a. Trim71 protein interactions are largely independent of Ago2 levels, but strongly depend on the presence of RNA. A second set of experiments is mass spectrometry data from immunoprecipitation experiments in mESCs overexpressing a tagged peptide derived from Homo sapiens TNRC6B (‘FLAG-HA::T6BWT::Cherry’), that is known to block the Tnrc6-Ago2 interaction. We show that a wild-type version strongly binds Ago1 and Ago2, but not Trim71, while a mutant version binds neither Ago1, 2, nor Trim71.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Daniel Hess

LAB HEAD: Helge Grosshans

PROVIDER: PXD034537 | Pride | 2023-07-04

REPOSITORIES: Pride

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Project description:Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cells (mESCs) proliferation. Using single pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. Nup153 is a large multifunctional protein that interacts with both the Y-complex and Tpr through its N-terminal NPC-targeting domain (NTD, [aa 1-339]) (Hase and Cordes, 2003; Vasu et al., 2001). To explore the possibility that the interaction between Nup153-NTD and the Y-complex or Tpr requires Nup133, we performed pull down assays on whole protein extracts from WT and Nup133-/- mESCs using recombinant Nup153-NTD [aa 1-339] as bait. Nup153 [aa 1-245], lacking the binding domains for the Y-complex and Tpr served as control. Western blot and quantitative mass spectrometry analyses detected Nup133 only in WT samples and documented its specific enrichment, along with other tested Y-Nups and Tpr, in the Nup153-NTD [aa 1-339] bound fraction. These analyses also revealed a similar enrichment of Tpr and Y-complex proteins in Nup153-NTD [aa 1-339]-purified extracts from either WT or Nup133-/- mESCs. These data show that in the context of whole cell lysates containing NPCs as dissociated subcomplexes, the lack of Nup133 neither precludes nor appreciably affects the biochemical interaction between Nup153-NTD and the Y-complex or Tpr. This differs from the in vivo situation that revealed a measurable impact of Nup133 on Nup153 dynamics and Tpr localization in the context of assembled NPCs. This apparent discrepancy between our in vitro and in vivo data indicates that the structural integrity of the NPC is likely required to visualize the contribution of Nup133 to the binding of Nup153 to Tpr or to the Y-complex. Hase, M.E., and Cordes, V.C. (2003). Direct interaction with nup153 mediates binding of Tpr to the periphery of the nuclear pore complex. Mol Biol Cell 14, 1923-1940. Vasu, S., Shah, S., Orjalo, A., Park, M., Fischer, W.H., and Forbes, D.J. (2001). Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export. J Cell Biol 155, 339-354.

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	2081_FT_S06.raw	Raw
	2081_W_S01.raw	Raw
	2081_W_S02.raw	Raw

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Convergence of distinct RNA-silencing pathways on GW182/Tnrc6

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