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. 2022 Sep 12;23(1):192.
doi: 10.1186/s13059-022-02759-y.

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation

Berta Duran-Arqué #  1 , Manuel Cañete #  1 , Chiara Lara Castellazzi  1 , Anna Bartomeu  1 , Anna Ferrer-Caelles  1 , Oscar Reina  1 , Adrià Caballé  1 , Marina Gay  1 , Gianluca Arauz-Garofalo  1 , Eulalia Belloc  1 , Raúl Mendez  2   3
Affiliations

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation

Berta Duran-Arqué et al. Genome Biol. .

Abstract

Background: Vertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3' UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date.

Results: Based on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2-4 are regulated by multiple proline-directed phosphorylations that control their liquid-liquid phase separation. CPEB2-4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs.

Conclusions: Altogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Keywords: 3′ UTR; BioID; CCR4-NOT complex; CPEB; Deadenylation; Phase separation; Phosphorylation; mRNA translation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CPEBs are co-expressed and co-regulated by phosphorylation in the meiotic cell cycle. A Endogenous CPEB2 and CPEB3 immunoblots from b-isox-precipitated extracts from the indicated meiotic maturation and early development time points. CPEB2, n = 2; CPEB3, n = 4. Vinculin immunoblot was used as a loading control. The number of oocytes loaded is indicated in parentheses. B Table showing the number of peptide spectrum matches (PSMs) of the CPEBs detected by MS/MS after b-isox-precipitation of meiotic maturation lysates, n = 1. C, D. Western blots of HA-CPEB2 (n = 2) and HA-CPEB3 (n = 2) overexpressing oocytes during meiotic maturation. Vinculin immunoblot was used as a loading control. E Lambda phosphatase assays (λ-PPase) of HA-CPEB2 (n = 2) and HA-CPEB3 (n = 2) overexpressing oocytes at the indicated time points. Western blots of anti-HA and anti-vinculin are shown. F CPEB3 phospho-to-total occurrence ratios determined by MS/MS for the indicated residue positions on prophase I (PI) versus progesterone-treated oocytes (+ Prog.). The ratios were calculated from the pool of 4 biological replicates. Only positions with a relative gain of phosphorylation upon progesterone treatment are displayed. Proline-directed sites are highlighted in bold. Error bars represent the ratio error. G Relative positions of the 18 proline-directed sites in CPEB3. The NTD is white-shaded, whereas the CTD is gray-shaded. H Wild-type (wt) and phosphomimetic (DE) CPEB3-NTD [γ-32P]-ATP incorporation upon incubation with oocyte lysates at the indicated maturation time points (n = 2). I Western blot against HA-tag and endogenous CPEB1 of stage VI ( −) and progesterone stimulated oocytes ( +) overexpressing wild-type HA-CPEB3 (wt), phosphonull HA-CPEB3 (A), and phosphomimetic HA-CPEB3 (DE). Not injected (NI) (n = 6). J Relative positions of the 20 proline-directed sites in CPEB2. The NTD is white-shaded, whereas the CTD is gray-shaded. K [γ-32P]-ATP incorporation by wild-type (wt) or phosphomutant (DE) CPEB2-NTD upon incubation with oocyte lysates at the indicated maturation time points (n = 3). L Western blot against HA-tag and CPEB1 of stage VI ( −) and progesterone stimulated oocytes ( +) overexpressing wild-type HA-CPEB2 (wt), not injected (NI), phosphonull HA-CPEB2 (A), and phosphomimetic HA-CPEB2 (DE) (n = 3). M The mean inhibition of [γ-32P]-ATP incorporation to CPEB2-NTD, CPEB3-NTD, or Histone H1 (H1) by increasing inhibitor concentrations. Data points represent the mean and standard deviation (n ≥ 3). Abbreviations: PI, prophase-I; MI, metaphase-I; MII, metaphase-II; hpf, hours post-fertilization; b-isox, biotinylated-isoxazole; λ-PPase, lambda phosphatase; In, input; Prog. or P, progesterone; DE, phosphomimetic mutant; A, phosphonull mutant; NTD, N-terminal domain; NL, no-lysate; NI, not-injected
Fig. 2
Fig. 2
The four CPEBs form cytoplasmic foci that possess distinct biophysical properties. A U-2 OS cells overexpressing either full-length or NTD CPEB1-4-GFP fusions. Scale bar, 10 μm. B Left: percentage of cells displaying “focal,” in blue, versus “diffuse,” in orange, cytoplasmic distribution of full length (FL) CPEB1-4 (1, 2, 3, 4), the latter defined by the absence of foci. Right: fold change (FC) increase in the number of “diffuse” cells in the N-terminal domain (NTD) constructs relative to full length (FL). CPEB1, n = 67; CPEB2, n = 72; CPEB3, n = 65; CPEB4, n = 67; CPEB1-NTD, n = 65; CPEB2-NTD, n = 65; CPEB3-NTD, n = 59; CPEB4-NTD, n = 63. C Quantification of CPEB1-4-GFP cytoplasmic foci features: sphericity, volume and number, and ratio soluble-to-total fluorescence intensity. n as specified for B. D The mean fluorescence recovery upon photobleaching (FRAP) curves of CPEB1-4-GFP. E Distribution of the half-time of recovery (t-half) and recovery fraction parameters obtained from the FRAP curves. CPEB1, n = 82; CPEB2, n = 95; CPEB3, n = 106; CPEB4, n = 109. F U-2 OS cells overexpressing either wt or phosphomimetic CPEB1-4-GFP fusions. Scale bar, 10 μm. G Fraction of cells displaying “focal,” in blue, versus “diffuse,” in orange, cytoplasmic distribution in wt versus phosphomimetic mutants. CPEB1 wt, n = 72; CPEB1-DE (6D-DD), n = 71; CPEB2 wt, n = 74; CPEB2-DE (20 DE), n = 83; CPEB3 wt, n = 65; CPEB3-DE (18 DE), n = 68; CPEB4 wt, n = 73; CPEB4-DE (12 DE), n = 72. In C and E, comparisons between the groups were carried out using the Kruskal–Wallis test (significance level 5%) and post hoc Dunn’s test with Holm’s correction. Significance scale: ****P-adj < 0.0001; ***P-adj < 0.001; **P-adj < 0.01; *P-adj < 0.05, non-significant differences not indicated. Abbreviations: FL, full-length; NTD: N-terminal domain; wt, wild-type; DE, phosphomimetic mutant
Fig. 3
Fig. 3
CPEB1 complex composition. A Western blots against the indicated proteins from several fractions of a gel-filtration fast protein liquid chromatography (GF-HPLC) done in PI-oocyte extracts. Approximate size of the fractions is noted above in KDa. B CPEB1 proximome in X. laevis PI oocytes, determined by BioID (n = 4). Hits include proteins enriched in CPEB1-BirA and BirA-CPEB1, relative to BirA alone. Hits meet the following criteria: either they have a positive fold change and P-adj < 0.05 relative to BirA alone or have 3 to 4 missing values in the control against 1 or none in the condition and a high relative abundance (greater than 25th percentile). C Co-immunoprecipitation of endogenous CPEB1 with the indicated HA-tagged baits (n = 2). D Co-immunoprecipitation of endogenous preys with HA-tagged CPEB1 (n = 3). The bands corresponding to PARN are indicated with arrows. E CPEB1, CPEB1(Y365A), and CPEB1-CTD proximomes determined by BioID in X. laevis PI oocytes (n = 4). The hits include the N-terminal and C-terminal BirA fusions and are defined as described in B. The size of the dots indicates the significance of the enrichment. F Co-immunoprecipitation of endogenous preys with HA-tagged CPEB1 and CPEB1(Y365A) (n = 2). G Co-immunoprecipitation of endogenous preys with HA-CPEB1 upon RNase A treatment (n = 3). H Co-immunoprecipitation of endogenous CPEB1 with the indicated HA-tagged baits upon RNase A treatment (n = 2). Abbreviations: I, input; E, elution; NI, not-injected; CTD, C-terminal domain. Indicated molecular weights are expressed in KDa
Fig. 4
Fig. 4
Comparative CPEB1-4 complex composition. A BioID-detected changes in the CPEB1(6A) proximome between PI (blue) and MII (yellow) (n = 3). The preys are compared in terms of their enrichment rank. Note that these ranks admit draws. B CPEB1-4 proximomes in X. laevis PI oocytes, determined by BioID (n = 4). Hits include proteins enriched in N-terminal and C-terminal BirA fusions, relative to BirA alone. Hits meet the following criteria: they either have a positive fold change and P-adj < 0.05 relative to BirA alone or have 3 to 4 missing values in the control against 1 or none in the condition and a high relative abundance (greater than 25.th percentile). C STRING network plot of the CPEB proximome in Xenopodinae displaying high confidence interactions (score > 0.7) and only networks with more than one interaction. CPEBs have been highlighted in yellow. D Co-immunoprecipitation of endogenous preys with HA-tagged CPEBs confirmed by Western blot using specific antibodies (n = 3)
Fig. 5
Fig. 5
CPEB1 and the CPEB2-4 subfamily target distinct mRNA subsets. A Western blot detection of HA and DDX6 of HA immunoprecipitates from HA-CPEB1-4 overexpressing oocytes (n = 3). The number of oocytes loaded is indicated in parentheses. I, input; E, eluate; NI: not-injected. B Overlap between the CPEB1-4 targets defined from RIP-Seq experiments. Targets are at least fourfold enriched relative to the input (P-adj ≤ 0.05) and twofold relative to the not-injected background control IP (P-adj ≤ 0.05). C Left: CPEB-mRNA enrichment heatmap for targets of at least one CPEB. The enrichment is expressed as the module of the two centered fold changes. The clustering tree was created with the full-linkage method. Right: CPEB1-CPEB2-4 differential enrichment heatmap. Colored genes are enriched at least twofold in one group versus the other (P-adj ≤ 0.05). D RIP-qPCR enrichment (expressed as delta CT) of indicated candidates in the CPEB1 IP relative to CPEB2/3/4 IPs. The candidates are either CPEB1-preferential targets or CPEB2-4-preferential targets, as indicated with dashed lines. Data points represent the mean and standard deviation (n = 3). E Motifs differentially enriched in the 3′ UTRs of targets of any CPEB-, CPEB1-, or CPEB2-4-preferential targets relative to RIP-Seq input minus targets and each group of preferential targets relative to the other, as determined with HOMER. The background used is indicated for the table rows. F AlphaScreen assay of purified CPEB1 and CPEB4 (50 nM) binding to CPE-A or CPE-G oligonucleotides. Error bars represent the standard deviation of the technical replicates (n = 2). The experiment was performed in triplicate. G Gene set enrichment in CPEB1-preferential targets (234 genes), CPEB2-4-preferential targets (414), or non-preferentially regulated targets (shared, 1148) determined with Enrichr. Only ontologies within “pathways” and “ontologies” with significant gene sets are included. Signaling by NOTCH1 (condensed) includes four redundant Reactome 2016 categories. GO MF, CC, and BP refer to molecular function, cellular component, and biological process, respectively. Significance scale: ****P-adj < 0.0001; ***P-adj < 0.001; **P-adj < 0.01; *P-adj < 0.05
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