Hwang H et al. (2022), Regulation of RNA localization during oocyte ma...

XB-ART-59529

Cell Rep 2022 Dec 13;4111:111802. doi: 10.1016/j.celrep.2022.111802.

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Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.

Hwang H , Yun S , Arcanjo RB , Divyanshi , Chen S , Mei W , Nowak RA , Kwon T , Yang J .

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Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

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Species referenced: Xenopus laevis
Genes referenced: cldn5 dnd1 esrp1 got1 hspa5 ocm psma1 psma2 psme3 psme4 ptbp1
GO keywords: oocyte maturation [+]
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	Graphical Abstract.
	Figure 1. Sorting of localized maternal transcripts during oocyte maturation (A and B) A schematic drawing shows the design of the experiments. RNAs were injected into the animal or vegetal poles of oocytes, respectively. Injected oocytes were cultured in either the OCM or OCM containing progesterone. Oocytes and mature eggs were dissected into animal and vegetal pieces for RNA extraction and subsequent qRT-PCR. The percentages of RNAs detected from each animal and vegetal pair were calculated. In (B), each black bar is 100%. These bars are placed at specific positions along the y axis of the graph to show the percentage distribution of RNAs along the animal/vegetal axis. Two-tailed Student’s t tests were performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n, number of animal and vegetal pairs being analyzed. (C) Confocal images show the distribution of Alexa 546-labeled psma1 and dnd1 RNAs in hemi-sectioned oocytes and mature eggs. White “∗” and “∧” label the animal and vegetal poles, respectively. (D) A schematic drawing shows the sorting of localized maternal transcripts during oocyte maturation.
	Figure 2. Remodeling of the ER during Xenopus OET (A–C) Immunofluorescence using an anti-KDEL antibody to stain the oocyte (A), egg (B), and two-cell-stage embryo (C). (A′, A″, B′, B″, C′, and C″) High-magnification images of the boxed areas in (A), (B), and (C), respectively. (D and E) Confocal images show the expression of GFP-KDEL in the oocyte (D and D′) and egg (E, E′, E″, and E‴). (D′) High-magnification image of the boxed area in (D). (E′, E″, and E‴) High magnification images of the boxed areas in (E). The position of GV in (A and D) is marked by “∗.” White arrowheads in (A) and (D) point to the dense ER patches associated with the GV. Blue arrows point to the animal subcortical region. Yellow arrows point to the ER in the vegetal cortex. Green arrowheads in (A″) highlight the long unbranched ER sheets around GV. Green arrows in (B″) point to three-way ER junctions. White arrowheads in (E′) and (E‴) point to the tubular ER that forms a network in the animal hemisphere of the egg.
	Figure 3. Association of proteasome mRNAs with the ER is dynamically regulated during Xenopus and mouse oocyte maturation (A) Confocal images show the distribution of Alexa 546-labeled psma1 RNA in GFP-KDEL overexpressed oocytes and eggs. Lower panel images are high-magnification views of the boxed area in the upper panel. (B) A schematic drawing shows the design of the fractionation experiments. (C) Agarose gel electrophoresis of RNAs extracted from the cytosolic, ER, and pellet fractions of oocytes and eggs. Arrows point to 18S and 28S rRNAs. (D) qRT-PCR results show the percentage distribution of psma1, psme1, psme2, psme3, and psme4 across the cytosolic, ER, and pellet fractions. The values of proteasome mRNAs were normalized to that of the spike-in gfp. The normalized values were used to calculate the percentage distribution of individual RNAs across three fractions. (E) Confocal images show the association of psma1 with the ER during mouse oocyte maturation. Mouse oocytes and ovulated eggs were analyzed by fluorescent in situ hybridization of psma1, in conjunction with immunofluorescence (IF) of GRP78. White circles in the upper panel mark the GV of the oocyte. (F) Representative images for cytosolic psma1 (upper panel) and ER-associated psma1 (lower panel). The left panels are high-magnification confocal images. The right panels show an analysis of psma1 and ER by ImageJ. (G) Bar graphs show the percentage of ER-associated psma1 in oocytes and ovulated eggs. Two-tailed Student’s t tests were performed. ∗p < 0.05; n, number of samples being analyzed.
	Figure 4. Transcriptome profiling analysis on the RNA localization associated with ER during oocyte maturation (A) A plot shows the absolute amount of RNAs (amol; top panels) and the proportion (percentage; bottom) of 14 20S proteasome α subunit mRNAs in each fraction (red, cytosol; green, ER; blue, pellet). The mean and standard errors of three biological replicates are presented. (B) Scatterplot shows the cytosol to ER ratio in the oocyte (y axis) and egg (x axis). Distribution plots (bottom, egg; left, oocyte) show the distribution of cytosol/ER ratios for all transcripts. Differentially localized transcripts were defined as the adjusted p value (q value) less than 0.05 and a greater than 2-fold difference between cytosol and egg (see also Figure S5). The yellow line represents an expected value for a transcript with no RNA localization change during oocyte maturation. Along both axes, the gray line shows the distribution of whole transcripts, and the pink line and red dots show the distribution of differentially localized transcripts. (C) Plots show the absolute amount and distribution of the top 100 differentially localized transcripts belonging to the oocyte-ER/egg-cytosol group in each fraction. (D) Distribution plot shows many transcripts were altered their localization during oocyte maturation, represented as a shift of the cytosol/ER ratio (dotted line, expected ratio for a transcript with no localization change). All-transcriptome profile was performed on biologically independent triplicated samples.
	Figure 5. Roles of ER-associated RBPs in regulating mRNA-ER association during oocyte maturation (A) Volcano plot shows the proteomic profiling of microsomes purified from oocytes and mature eggs. Differentially expressed proteins were defined as a p value less than 0.05 (dashed horizontal line) and greater than a 2-fold difference between oocyte and egg (dashed vertical lines) (gray dots, whole 1,976 proteins; black dots, RBPs). All-proteomic profile was performed on biologically independent triplicated samples. (B) Bar graph shows gene ontology (GO) enrichment analysis of ER-associated proteins that remain unchanged during oocyte maturation. This analysis was performed by Metascape. (C) Western blot shows the expression of RBPs (Ptbp1, HuR, and Tia1) in cytosolic, ER, and pellet fractions in the oocyte and mature egg. Atlastin and Actin were enriched in the ER and cytosolic fractions, respectively. (D) Pie charts show the summary of RIP assays. In all three RIP assays, 23 transcripts in the oocyte-ER/egg-ER groups and 23 transcripts in the oocyte-ER/egg-cytosol groups were analyzed (pink, the proportion of mRNAs with decreased binding; gray, the proportion of mRNAs with no change; blue, the proportion of mRNAs with increased binding; yellow, the proportion of mRNAs not bound by RBPs). (E) Bar graphs are Ptbp1-RIP of cldn5, esrp1, and psap, which are shown as representative results of the increased, decreased, and unchanged categories, respectively.
	Figure 6. Interfering with the tubular ER network via dissociation of F-actin impairs the localization of proteasome mRNAs during oocyte maturation (A–D) Confocal images of phalloidin-stained oocytes and eggs. White boxes indicate the areas where high-magnification images were taken from. Arrowheads point to F-actin in control oocytes and eggs. (E–H) Confocal images of GRP78 staining show the morphology of the ER network in control and F-actin inhibitor-treated samples. (I) In situ hybridization results show the effects of F-actin inhibitors on proteasome mRNA distribution in oocytes and eggs. Arrows mark the lower edge of the proteasome mRNA expression domain in the equator of mature eggs. CB, cytochalasin B; n, number of samples being analyzed.
	Figure 7. Enucleation disrupts ER remodeling and proteasome mRNA localization during oocyte maturation (A) Images show the process of enucleation. Arrowheads point to the GV. (B) Confocal images of anti-KDEL antibody staining, showing that enucleated oocytes failed to develop an ER network in the animal hemisphere after progesterone treatment. (C) Anti-KDEL-positive signals in images of (B) were quantified using ImageJ. Two-tailed Student’s t tests were performed. ∗p < 0.05, ∗∗p < 0.01; n, number of samples being analyzed. (D) In situ hybridization shows that enucleation disrupted animal localization of proteasome mRNAs after oocyte maturation. The number of samples exhibiting the phenotype and the sample size is provided at the lower right corner of each figure.
	S-Fig 1. ER dynamics during the OET. Related to Figure 2. A. In situ hybridization of a hemi-sectioned egg, showing the expression of psma1. White arrowheads marked the lower edge of the pigmented area on the surface of the animal hemisphere. B and C. Histology of a fully-grown oocyte (B) and egg (C). Dashed lines mark the upper border of the heavy yolk platelet-rich territory in the egg. Inserted at the lower right corner are high-magnification images of the boxed areas in the equatorial region. D – I. Representative TEM images from the animal (D, E, G, H) and vegetal (F, I) hemispheres of an oocyte (D, E, F) and an egg (G, H, I). White arrows (G) and circle (H) highlight a small cluster of tubular ER and a dense ER patch in the animal hemisphere of an egg, respectively. H’ is an enlarged image of the boxed area in H. J – O. Immunofluorescence staining of pan-Atlastin (J, K, L) and GRP78 (M, N, O) in oocytes (J, M), eggs (K, N), and 2-cell stage embryos (L, O). The position of GV in J and M was marked by “*”. White arrowheads in M point to dense ER patches associated with GV.
	S-Fig 2. Association between proteasome mRNAs and the ER during oogenesis and early development. Related to Figure 3. A and D. Immunofluorescence staining of KDEL shows the distribution of the ER in stage I (A) and stage II/III (D) oocytes. B, C, E, F-J. In situ hybridization of hemi-sectioned oocytes, showing the expression of psma1 and psme3 in stage I (B and C), stage II/III (E and F), stage VI (G and H) oocytes, and mature eggs (I and J). All these samples were stained in BM-purple for 3 hours. Inserts at the lower left corner in G, H, I, and J are samples being stained in BM-purple overnight. K-N. Fractionation RT-qPCR results show the percentage distribution of psma1 (K and L), and psme3 (M and N) across the cytosolic, ER, and pellet fractions during oogenesis (K and M) and early development (L and N).
	S-Fig 3. Western blot shows efficient fractionation of Xenopus oocytes and eggs. Related to Figure 3. Fractionated samples were subjected to western blot analysis, using anti-pan-Atlastin and anti-Hsc70 antibodies. Results show that Atlastin and Hsc70 were enriched in the ER and cytosolic fractions, respectively.
	S-Fig 4. Transcriptomic quantification in the cell fractionation for the mRNA-ER association analysis during oocyte maturation. Related to Figure 4. A. The graph shows the linear correlation of the RNA-seq estimated TPM values and the actual input amount of five spike-in RNAs in the oocyte and mature egg. The linear regression of them was used to convert the TPM to the absolute atto-moles (amol). B. The bar graph shows the distribution of total RNAs in each cytosolic, ER, and pellet fraction between oocyte and mature egg. We normalized the total amount of RNAs for each sample (we used the same number of eggs and oocytes) presented on the right side. C. Histogram shows the distribution of the mRNA absolute amount in the oocyte (blue series) and mature egg (red series). The dashed line indicates 0.125 amol, and transcripts less than 0.125 amol were filtered out. A total of 17,811 transcripts were used for further analysis. D-E. Hierarchical clustering dendrogram (D) and principal component analysis (PCA) plot (E) of three biological replicates in the oocyte and mature egg, respectively. F. Histogram shows the distribution of the proportion of transcripts in each cytosolic (pink), ER (blue), and pellet (grey) fraction in the oocyte (top) and mature egg (bottom).
	S-Fig 5. Down-regulation of the mRNA-ER association during oocyte maturation. Related to Figure 4. A. B. MA plots show the difference in the ratio of the cytosol to ER between the oocyte (A) and mature egg (B). The xaxis indicates the amount of RNAs, and the y-axis indicates the ratio of transcripts between the cytosol and ER. A differentially localized gene (red) was defined as a q-value less than 0.05 and cytosol to ER ratio difference greater than 2 (Grey - total 17,811 transcripts). 4,179 and 432 transcripts in the oocyte are significantly biased to the cytosol and ER, respectively. However, these numbers were more skewed in the mature egg, as 16,491 transcripts in the cytosol and 37 in the ER were differentially localized.
	S-Fig 6. Validation of fractionation RNA-seq. Related to Figure 4. The percentage distribution of psma1, nme3.S got1.S, mprip.S, snph.S, spata13.S, sptan1.S, and capn5.L across the cytosolic, ER, and pellet fractions were calculated using the RNA-seq data and plotted into graphs in the upper panel. The middle panel is RT-qPCR validation of the distribution of the above transcripts using RNAs purified from the detergent-based fractionation. The lower panel is RT-qPCR validation of the ER association of the above transcripts using RNAs purified from microsomes. Purification of microsomes was carried out using the standard sucrose density gradient ultracentrifugation protocol. Among all transcripts being tested, only psma1.S, nme3.S, and got1.S show a decreased ER association after oocyte maturation. RT-qPCR was performed on biologically independent triplicated samples and Two-tailed Students’ t-tests were performed. p<0.05; *p<0.01.
	S-Fig 7. The majority of transcripts exhibiting an increase in the polysome fraction during oocyte maturation are released from the ER into the cytosol. Related to Figure 4. Transcripts showing a significant increase in the polysome fraction during oocyte maturation are plotted in the MA plots to show the difference in the ratio of the cytosol to ER in the oocyte (left) and mature egg (right). The x-axis indicates the amount of RNAs, and the y-axis indicates the ratio of transcripts between the cytosol and ER. Transcripts being significantly released from the ER into the cytosol are shown in black. Red dots represent transcripts whose ER association remains unchanged.

References [+] :

Aguero, Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex. 2017, Pubmed, Xenbase