Lavering ED et al. (2023), Intrinsically disordered regions are not suffic...

XB-ART-60404

PLoS Biol 2023 Nov 01;2111:e3002378. doi: 10.1371/journal.pbio.3002378.

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Intrinsically disordered regions are not sufficient to direct the compartmental localization of nucleolar proteins in the nucleus.

Lavering ED , Gandhamaneni M , Weeks DL .

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The nucleolus is a non-membrane bound organelle central to ribosome biogenesis. The nucleolus contains a mix of proteins and RNA and has 3 known nucleolar compartments: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The spatial organization of the nucleolus is influenced by the phase separation properties of nucleolar proteins, the presence of RNA, protein modification, and cellular activity. Many nucleolar proteins appear to concentrate within the borders of the compartments. We investigated whether the intrinsically disordered regions from several proteins provided the information needed to establish specific compartment localization using Xenopus laevis oocytes. For the proteins we tested, the disordered regions were not sufficient to direct specific domain localization and appear dispensable with respect to compartmentalization. Among the proteins that colocalize to the DFC are the quartet that comprise the box H/ACA pseudouridylation complex. In contrast to the insufficiency of IDRs to direct compartment localization, we found that the DFC accumulation of 2 box H/ACA proteins, Gar1 and Nhp2, was disrupted by mutations that were previously shown to reduce their ability to join the box H/ACA complex. Using a nanobody to introduce novel binding to a different DFC localized protein, we restored the localization of the mutated forms of Gar1 and Nhp2.

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Species referenced: Xenopus laevis
Genes referenced: bop1 dkc1 fbl gar1 gtpbp4 ncl nhp2 nop56 npm1 npm3 pak1ip1 pes1 rpl12
GO keywords: fibrillar center [+]

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	Fig 1. IDRs in nucleolar localization. (A) Model of the 3 nucleolar subdomains or compartments. (B) Biological variation of wild-type Ncl-GFP co-expressed with Fbl-mRed, shown as expressed in four different frogs, showing that Ncl is always in the GC and sometimes spreads into the DFC. (C) Schematic of the protein fusions shown in D-F. Each of these fusions also contains a green or red fluorescent protein as indicated in B that allowed detection. The IDR-Ncl has a net negative charge of −40, the IDR-Fbl has a net positive charge of +17, and the N terminal IDR-Gar1 has a net positive charge of +7. (D–F) Representative images of nucleoli with IDR chimera proteins fused with a fluorescent protein co-expressed with a DFC nucleolar domain marker (Fbl). (D) Representative images showing the intrinsically disordered regions of Fbl (IDR-Fbl-GFP) and Ncl (IDR-Ncl-GFP) co-expressed with Fbl-mRed. (E) Representative images showing the fusions of IDR-Ncl and Fbl, showing IDR-Ncl-Fbl-eGFP, IDR-Ncl-ΔNFbl-eGFP, and ΔNFbl-eGFP all co-expressed with Fbl-mRed. *IDR-Ncl-Fbl-eGFP images were taken at a lower exposure in the green channel due to it being far brighter than the other constructs in this set. (F) Representative images showing the fusions of IDR-Ncl and Gar1, showing the localization of Gar1-mCherry (WT that localizes to the DFC), IDR-Ncl-Gar1-mCherry, IDR-Ncl-ΔNGar1-mCherry, and ΔNGar1-mCherry all co-expressed with Fbl-eGFP. Scale 10 μm. Statistical analysis is shown in S1 Fig and S2 Data. DFC, dense fibrillar component; IDR, intrinsically disordered region.
	Fig 2. Nucleolar localization after binding disruption. (A) Model of the general layout of the box H/ACA complex proteins and a schematic showing the mutation sites to disrupt binding. (B) Representative images of nucleoli expressing wild-type Gar1-mCherry and Gar1 with a mutated binding site to DKC1 (Gar1M1-mCherry) along with the DFC marker Fbl-eGFP. Also shown are wild-type Nhp2-mRed and Nhp2 with a point mutation that disrupts its binding to Nop10 (Nhp2P83A-mRed) with DFC marker Fbl-eGFP. Isolated nuclei were incubated in OR2 for 20 min prior to imaging. Scale: 10 μm. (C) Quantification of the colocalization of Gar1M1-mCherry or Gar1-mCherry with Fbl-eGFP and of Nhp2-RFP or Nhp2P83A-RFP with Fbl-eGFP shown with Pearson’s coefficient. Significance was determined using a t test (p < 0.05) with N = 30 (Gar1) and 26 (Gar1M1) for the Gar1 set and N = 24 (Nhp2) and 21 (Nhp2P83A) for the Nhp2 set. These experiments were repeated with nucleoli from the oocytes of 3 different frogs and were significant each time. The underlying data can be found in S1 Data. (D) Images of Nhp2-mRed and Nhp2P83A-mRed showing extra-nucleolar deposits of Nhp2P83A (arrows) that were not present for the wild type. Scale: 20 μm. DFC, dense fibrillar component.
	Fig 3. Nanobody binding can induce DFC localization. (A) Representative nucleoli from oocytes expressing Gar1 and Gar1M1 nucleolar localization and the same proteins fused with a GFP nanobody (Nb). These proteins were all fused with a red fluorescent protein and were co-expressed with Fbl-eGFP, a DFC marker. (B) Nhp2 and Nhp2P83A with and without nanobody fusion co-expressed with Fbl-eGFP. (C) The localization of a monomeric fluorescent protein (mRed) and Npm1 bound to the GFP nanobody, co-expressed with Fbl-eGFP. For each condition in Fig 3, 35+ nucleoli were observed from each of 3 different injection days, representative images are shown here. Scale: 20 μm. DFC, dense fibrillar component.
	Fig 4. Nuclear fusion experiments. (A) Schematic of nuclear fusion experiments, showing nuclei being isolated in mineral oil; 2 nuclei, each expressing different fluorescently tagged proteins, being fused together; then fused nuclei being added to a microscope slide in a well of petroleum jelly for imaging. (B) Fluorescent images taken in 10-min intervals showing the diffusion of the fluorescently tagged proteins from 1 nucleus to the nucleus it is fused with. The top row shows a nucleus containing Fbl-eGFP fused with a nucleus containing Npm1-mRed. The second row shows a nucleus with Fbl-eGFP fused with a nucleus expressing Npm1-mRed-Nb. Scale: 50 μm. (C) Representative Apotome fluorescent images of nuclei found at the junction of the fused nuclei after 40 min for the proteins indicated on each image. For each condition in Fig 4, 35+ nucleoli were observed from each of 3 different injection days, representative images are shown here. Scale: 20 μm.
	S1 Fig. Statistical analysis for Fig 1. (A) Statistical analysis using Pearson’s Coefficient to assess the overlap of fluorescently tagged Ncl co-expressed with fluorescently tagged Fbl on 4 different days using one-way ANOVA multiple comparisons test, p < 0.05. N = 15, 58, 52, and 24 for days 1–4 respectively. (B) Table showing the results of the statistical analysis comparing the coefficients of variations of the conditions indicated in the left column. Replicates “1, 2, and 3” indicate different frogs/injection days for each condition. Values from columns are not necessarily from the same day (data should be read horizontally and not vertically). P < 0.05, N values between 15–62. The underlying data can be found in S2 Data.
	S2 Fig. Response of Fbl and Gar1 to 1,6-hexanediol treatment. (A) Representative images of nucleoli expressing Gar1-GFP and Fbl-mRed from nuclei that were isolated in OR2 and soaked in 10% 1,6-hexanediol for 10 min. (B) Analysis of co-localization using Pearson’s coefficient between Gar1 and Fbl with and without hexanediol (p < 0.05) N = 35 (control) and 33 (hexanediol). This experiment was repeated with nucleoli from the oocytes of 3 different frogs and with the fluorescent tags switched. The underlying data can be found in S3 Data.

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