Neil CR et al. (2021), L-bodies are RNA-protein condensates driving RN...

XB-ART-58511

Mol Biol Cell 2021 Dec 01;3222:ar37. doi: 10.1091/mbc.E21-03-0146-T.

Show Gene links Show Anatomy links

L-bodies are RNA-protein condensates driving RNA localization in Xenopus oocytes.

Neil CR , Jeschonek SP , Cabral SE , O'Connell LC , Powrie EA , Otis JP , Wood TR , Mowry KL .

???displayArticle.abstract???
Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

???displayArticle.pubmedLink??? 34613784
???displayArticle.link??? Mol Biol Cell
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: cpeb1 dnai1 gapdh gdf1 hnrnpab igf2bp3 isyna1 kif3a lrwd1 lsm14b lsm2 mtor nos1 padi2 pmch stau1 trim36 vegt ybx1
GO keywords: P-body [+]
???displayArticle.antibodies??? Tuba4b Ab2

???attribute.lit??? ???displayArticles.show???

	FIGURE 1: Vegetally localizing mRNA is contained in a large RNP complex. (A) A cryosection of a stage II oocyte probed by FISH for vg1 mRNA (magenta) and vegT mRNA (A′, green) is shown, with the vegetal cortex at the bottom. Scale bar = 50 µm. (B) Higher magnification view of the vegetal cytoplasm of a stage II oocyte probed by FISH for vg1 mRNA (magenta), shown merged in B′ with vegT mRNA (green). Scale bar = 10 µm. (C) A cryosection of a stage II oocyte probed by FISH for vg1 mRNA (magenta) and gapdh mRNA (C′, green) is shown, with the vegetal cortex at the bottom. Scale bar = 50 µm. (D) Higher magnification view of gapdh mRNA in the vegetal cytoplasm of a stage II oocyte probed by FISH for gapdh mRNA (green), shown merged in D′ with vg1 mRNA (magenta). Scale bar = 10 µm. (E) A cryosection of a stage II oocyte probed by FISH for vg1 mRNA (magenta) nos1 mRNA (E′, green), and merged in E′′ is shown. The vegetal cortex is at the bottom. Scale bar = 50 µm. (F) Higher magnification view of nos1 mRNA (green) and vg1 mRNA (magenta) in the vegetal cytoplasm of a stage II oocyte. The vegetal cortex is at the bottom; scale bar = 10 µm. (G) A cryosection of a stage II oocyte is shown, with the vegetal cortex at the bottom. Combined FISH-IF was used to detect vg1 mRNA (magenta) and Stau1 protein (G′, green). Scale bar = 50 µm. (H) Combined FISH-IF was used to detect Stau1 (H, green) in the vegetal cytoplasm of a stage II oocyte, merged with vg1 mRNA (magenta) in H′. Scale bar = 20 µm. (I) High magnification view of the vegetal cytoplasm of a stage II oocyte showing colocalization of vg1 mRNA (magenta) and Stau1 protein (green). Scale bar = 10 µm. (J) A cryosection of a stage II oocyte is shown with the vegetal cortex at the bottom. Combined FISH-IF was used to detect vg1 mRNA (magenta) and hnRNPAB protein (J′, green). Scale bar = 50 µm. (K) Combined FISH-IF was used to detect hnRNPAB (K, green) in the vegetal cytoplasm of a stage II oocyte, merged with vg1 mRNA (magenta) in K′. Scale bar = 20 µm. (L) High magnification view of the vegetal cytoplasm of a stage II oocyte showing colocalization of vg1 mRNA (magenta) and hnRNPAB protein (green). Scale bar = 10 µm.
	FIGURE 2: Assembly into L-bodies is correlated with vegetal localization. (A) A cryosection of a stage II oocyte is shown, with microinjected fluorescently labeled LE RNA (green) and endogenous vg1 mRNA (A′) detected by FISH (magenta). The vegetal cortex at the bottom, and the scale bar = 50 µm. (B) High magnification view of the vegetal cytoplasm of a stage II oocyte with endogenous vg1 mRNA (magenta) detected by FISH and microinjected LE RNA (green) shown merged in B′. Scale bar = 10 µm. (C) A cryosection of a stage II oocyte is shown, with microinjected fluorescently labeled mutant LE RNA (mutLE) in green and endogenous vg1 mRNA (C′) detected by FISH in magenta. The vegetal cortex at the bottom and the scale bar = 50 µm. (D) High magnification view of the vegetal cytoplasm of a stage II oocyte with endogenous vg1 mRNA (magenta) detected by FISH and microinjected mutLE RNA (green) shown merged in D′. Scale bar = 10 µm.
	FIGURE 3: Isolation of L-bodies. (A) Schematic of the L-body isolation procedure. Stage II/III oocytes were formaldehyde cross-linked, homogenized, clarified by centrifugation, and fractionated by SE chromatography. Fractions containing vg1 mRNA were pooled and divided equally for immunoprecipitations using anti-Stau1 (Stau) and anti-hnRNPAB (AB) antibodies, with IgG as a negative control. (B) Stage II–III oocytes were microinjected with LE RNA (green), nonlocalizing mutLE RNA (red), and nonlocalizing β-globin RNA (blue). Oocyte lysates were subjected to SE chromatography and the levels of the microinjected RNAs and endogenous vg1 mRNA (black) were measured in SE column fractions by RT-qPCR, normalized to luciferase control RNA (luc). Error bars represent standard error of the mean (SEM); n = 4 columns. (C) Oocyte lysate was treated with RNase A prior to SE chromatography, followed by immunoblot analysis with anti-Vera and anti-Tubulin antibodies. Column fractions from untreated (–) lysate are shown at the top and column fractions from RNase-treated (+) lysate are shown at the bottom. (D) Immunoblot analysis is shown, of SE column fractions probed with anti-Stau1, anti-hnRNPAB, and anti-Tubulin. The input (load) is at the left and fraction numbers are shown at the bottom. Stau1 and hnRNPAB chromatograph primarily in the void volume; fractions 2–5, which were pooled for further purification. (E) Pooled SE column fractions (2–5) were immunoprecipitated (IPed) using anti-Vera, anti-Stau1, anti-hnRNPAB, and IgG. Following isolation of bound RNA, vg1 RNA was detected by RT-qPCR, with normalization to a luciferase RNA control. Shown is log2-fold enrichment for vg1 RNA from the Vera, Stau1, and hnRNPAB co-IPs over IgG. n = 5 and error bars represent SEM. *p < 0.01, p < 0.05. (F) Pooled SE column fractions (2–5) were IPed using anti-Vera (V), anti-Stau1 (S), anti-hnRNPAB (AB), and IgG. After SDS–PAGE, co-IP of Vera, Stau1, and hnRNPAB were confirmed by immunoblotting with anti-Vera, anti-Stau1, and anti-hnRNPAB. (G) RNA-seq was performed on parallel IPs using anti-Stau1, anti-hnRNPAB, and IgG. A Venn diagram shows the overlap between hnRNPAB (magenta) and Stau1 (blue); 454 RNAs were identified as enriched over IgG in both the hnRNPAB and Stau1 IPs. See also Tables S1-S4. (H) MS was performed on parallel IPs using anti-Stau1, anti-hnRNPAB, and IgG. A Venn diagram shows the overlap between hnRNPAB (magenta) and Stau1 (blue); 86 proteins were identified as significantly enriched over IgG in both the hnRNPAB and Stau1 IPs.
	FIGURE 4: L-bodies contain protein components common to other classes of cytoplasmic RNP granules. (A) Protein constituents of L-bodies are also found in stress granules (S, orange), P-bodies (P, green), neuronal granules (N, yellow), and germ granules (G, blue). Proteins found in L-bodies but not reported in other granules, are italicized and previously known vg1 RBPs are bolded. (B–I) IF and combined FISH-IF were performed to validate potential protein constituents of L-bodies. (B, C) Shown are IF images of stage II oocytes using (B) anti-Ddx3, (C) anti-Lsm2, (D) anti-Lsm14B, and (E) anti-Ybx1. The vegetal cortex is at the bottom; scale bars = 20 µm. (F–I) Shown are FISH-IF images of the vegetal cytoplasm of stage II oocytes with the vegetal cortex at the bottom; scale bars = 10 µm. Shown in magenta is vg1 mRNA detected by FISH. Merged in green is IF using (F) anti-Ddx3, (G) anti-Lsm2, (H) anti-Lsm14B, and (I) anti-Ybx1. See also Supplemental Table S5. (J) Immunoblot analysis is shown of SE column fractions probed using anti-Cpeb1, anti-Ddx3, anti-G3bp2, anti-Pabc1, anti-Ybx1, and anti-Tubulin antibodies. The fraction numbers are indicated at the bottom. (K) Overlap (black, 73.3%) of L-body constituents with other cytoplasmic granules; unique proteins are indicated by gray (26.7%). (L) The percentage of identified L-body proteins that are found in other cytoplasmic RNP granule types: stress granules (59.3%, 51 of 86), P-bodies (23.3%, 20 of 86), neuronal granules (34.9%, 30 of 86), and germinal granules (10.5%, 9 of 86). See also Supplemental Table S7.
	FIGURE 5: L-bodies exhibit a meshlike structure. (A) Comparison by SLIDER analysis (Peng et al., 2014 blue right-pointing triangle) for long IDRs (>30 consecutively disordered residues) for the X. laevis proteome vs. the L-body proteome. *p < 0.001. (B) The percentage of prionlike domains in the L-body proteome was compared with the Xenopus proteome using PLAAC. Prionlike domains are sixfold enriched in L-bodies (12.9%) relative to the Xenopus proteome (2.02%). *p < 0.001. (C) A stage II oocyte stained with thioflavin is shown, with the vegetal cortex at the bottom. Scale bar = 50 µm. (D) High magnification view of the vegetal cytoplasm of a stage II oocyte stained with thioflavin. Scale bar = 10 µm. (E–G) High magnification views of the vegetal cytoplasm of stage II oocytes stained with thioflavin are shown; scale bars = 10 µm. Thioflavin staining (green) was combined with FISH (magenta) for detection of the following RNAs: (E) vg1, (F) vegT, and (G) gapdh.
	FIGURE 6: L-bodies contain dynamic proteins and nondynamic localized RNAs. (A) Stage II oocytes expressing mCh, or the following mCh-tagged proteins: hnRNPAB, Ybx1, Stau1, and Vera, were microinjected with Cy5-labeled vg1 LE RNA to mark L-bodies. Recovery curves are shown for mCh (red), mCh-hnRNPAB (blue), Ybx1-mCh (orange), Stau1-mCh (green), and Vera-mCh (purple). Error bars indicate standard error of the mean (SEM). (B) An image of the vegetal cytoplasm of an oocyte microinjected with mCh-hnRNPAB is shown, with a 10 µm2 ROI (yellow); scale bar = 10 µm. B′ and B′′ show the postbleach and 500 s time points, respectively. Photobleaching was corrected using the ImageJ plugin CorrectBleach V2.0.2. Zenodo (Miura et al., 2014 blue right-pointing triangle) (C) Stage II oocytes were microinjected with Cy3-labeled vg1 LE RNA to mark L-bodies and coinjected with the following Cy5-labeled RNAs for FRAP analysis: gapdh, mutated vg1 LE (mutLE), vegT LE (TLE), or vg1 LE (LE). Recovery curves are shown for gapdh RNA (blue), mutLE RNA (red), TLE RNA (gray), and LE RNA (green). Labeling of RNAs with Cy3 vs. Cy5 does not affect dynamics (see Figure S4). Error bars show SEM. (D) An image of the vegetal cytoplasm of an oocyte microinjected with Cy5-labeled LE RNA is shown, with a 10 µm2 ROI (yellow); scale bar = 10 µm. D′ and D′′ show the postbleach and 500 s time points, respectively. Photobleaching was corrected using the ImageJ plugin CorrectBleach V2.0.2. Zenodo (Miura et al., 2014 blue right-pointing triangle) (E) The mobile fraction is shown for LE RNA (green, 4.8 ± 0.6%), TLE RNA (gray, 19.2 ± 1.4%), mutLE RNA (red, 61.2 ± 3.6%), and gapdh RNA (blue, 56.1 ± 6.7%), determined by FRAP as in panel C. The results from 7 oocytes per RNA are shown and error bars show SEM. P-values were calculated using one-way ANOVA with Tukey’s multiple comparison correction; *p < 0.001, ns is not significant. (F) The mobile fraction for mCh-hnRNPAB (blue, 94.2 ± 1.9%), Ybx1-mCh (orange, 80.1 ± 3.2%), Stau1-mCh (dark green, 74.5 ± 2.0%), Vera-mCh (purple, 52.9 ± 3.4%), TLE RNA (gray, 19.2 ± 1.4%), and LE RNA (green, 4.8 ± 0.6%) was determined by FRAP, as in panels A and C. The results from seven oocytes per protein and RNA are shown and error bars indicate SEM. P values were calculated using one-way ANOVA with Tukey’s multiple comparison correction; *p < 0.001, ns is not significant.
	FIGURE 7: Model for L-body structure and assembly. Localizing RNAs (blue) interact with specific RNA-binding proteins (RBPs; red) that facilitate local enrichment. Nonlocalizing RNAs (gold) lack binding sites for these RBPs and do not condense with other localized RNAs. Specific RBPs facilitate enrichment (large arrows) of localized RNAs in L-bodies (gray). L-body enrichment results in high local RNA concentrations, facilitating RNA–RNA, RNA–protein, and protein–protein interactions and formation of a nondynamic RNA phase enmeshed in a more dynamic protein phase. Nonlocalized RNAs (and proteins) are not excluded from L-bodies, and can freely move in and out (double arrows).
	Figure S1. Trim36 mRNA is enriched in L-bodies. Trim36 mRNA (Cuykendall and Houston, 2009), which is transported to the vegetal hemisphere during stages II-II of oogenesis, is contained in L-bodies. (A) A cryosection of a stage II oocyte probed by FISH for trim36 mRNA (green), shown merged in A¢ with vg1 mRNA (magenta) is shown. The vegetal cortex at the bottom. Scale bar=50 μm. (B) Higher magnification view of trim36 mRNA (green) and vg1 mRNA (magenta) in the vegetal cytoplasm of a stage II oocyte. The vegetal cortex is at the bottom; scale bar=10 μm.
	Figure S2. Vera protein is enriched in L-bodies. (A) Combined FISH-IF was used to detect vera protein (A, green) in the vegetal cytoplasm of a stage II oocyte, merged with vg1 mRNA (magenta) in A¢. The vegetal cortex is at the bottom; scale bar=50 μm. (B) High magnification view of the vegetal cytoplasm of a stage II oocyte showing colocalization of vg1 mRNA (magenta) and vera protein (green). Scale bar=10 μm.
	Figure S2. Vera protein is enriched in L-bodies. (A) Combined FISH-IF was used to detect vera protein (A, green) in the vegetal cytoplasm of a stage II oocyte, merged with vg1 mRNA (magenta) in A¢. The vegetal cortex is at the bottom; scale bar=50 μm. (B) High magnification view of the vegetal cytoplasm of a stage II oocyte showing colocalization of vg1 mRNA (magenta) and vera protein (green). Scale bar=10 μm.
	Figure S3. Xenopus oocyte RNA stains with thioflavin and phase-separates in vitro. (A) DIC Image of droplets formed at 1 mg/mL oocyte RNA. Scale bar=5μm. RNA droplets were imaged at room temperature with a Zeiss Axiovert 200M Fluorescent Microscope using a 100 ́ oil Plan- NEOFLUAR objective (NA=1.3). Images were taken in the AxioVision 4.8 software on a Hamamatsu ORCA-ER digital camera. (B) Image of RNA gel formed at 10 mg/mL Xenopus oocyte RNA. Scale bar=1mm. (C) Stage II-III Xenopus oocyte RNA was serially diluted into buffer containing 125 nM thioflavin-T (in 10 mM Tris pH=7.4, 4 mM MgCl2). After heating to denature RNA secondary structure, thioflavin fluorescence was monitored at 482 nm. RNA concentrations were as follows: 0.0625 (yellow), 0.125 (purple), 0.25 (green), 0.5 (blue), and 1 (red) mg/mL.
	Figure S3. Xenopus oocyte RNA stains with thioflavin and phase-separates in vitro. (A) DIC Image of droplets formed at 1 mg/mL oocyte RNA. Scale bar=5μm. RNA droplets were imaged at room temperature with a Zeiss Axiovert 200M Fluorescent Microscope using a 100 ́ oil Plan- NEOFLUAR objective (NA=1.3). Images were taken in the AxioVision 4.8 software on a Hamamatsu ORCA-ER digital camera. (B) Image of RNA gel formed at 10 mg/mL Xenopus oocyte RNA. Scale bar=1mm. (C) Stage II-III Xenopus oocyte RNA was serially diluted into buffer containing 125 nM thioflavin-T (in 10 mM Tris pH=7.4, 4 mM MgCl2). After heating to denature RNA secondary structure, thioflavin fluorescence was monitored at 482 nm. RNA concentrations were as follows: 0.0625 (yellow), 0.125 (purple), 0.25 (green), 0.5 (blue), and 1 (red) mg/mL.

References [+] :

Ahsan, Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction. 2017, Pubmed