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Dev Cell
2024 Apr 22;598:1058-1074.e11. doi: 10.1016/j.devcel.2024.02.007.
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Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos.
Xiang K
,
Ly J
,
Bartel DP
.
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During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.
Figure 1. A core CPE controls cytoplasmic polyadenylation in frog oocytes
(A) The N60-PASmos mRNA library.
(B) Experimental scheme for mRNA library injection and sample collection.
(C) Tail-length changes associated with each 5-mer in the 3′ UTRs of the N60-PASmos library, comparing between 0 and 7 h post-progesterone treatment. Plotted for each 5-mer are differences in mean tail lengths observed for mRNAs with that 5-mer.
(D) As in (C), but for 6-mers.
(E) Sequence logo generated from the 180 8-mers most strongly associated with tail lengthening in the N60-PASmos library, comparing between 0 and 7 h post-progesterone treatment.
(F) ROC curves testing the ability of previously proposed CPEs to classify endogenous mRNAs as subject to cytoplasmic polyadenylation during frog oocyte maturation. A curve is shown for each of the indicated CPEs, including the two canonical CPEs (UUUUAU and UUUUAAU), as well as for the indicated combinations of elements.
(G) ROC curves testing the ability of the best 3-, 4-, 5-, 6-, 7-, and 8-mers to classify endogenous mRNAs as subject to cytoplasmic polyadenylation during frog oocyte maturation.
(H) The effects of 5-mers on tail lengths of mRNAs in the N60-PASmos library during frog oocyte maturation. Plotted for each 5-mer are the mean tail lengths of mRNAs containing that 5-mer.
(I) As in (H), except mRNAs that contained UUUUA were excluded.
See also Figure S1.
Figure 2. Context of the CPE and the PAS influences cytoplasmic polyadenylation in frog oocytes
(A) Effect of the distance between the CPE and the PAS. Plotted for each of the four motifs is the difference in mean tail length observed for mRNAs of the N60-PASmos library (schematic at top) containing that motif at each position along the variable region of the 3′ UTR, comparing between 0 and 7 h post-progesterone treatment. Shaded areas along lines indicate standard error of the difference between means.
(B) Effect of the relative position of the CPE and the PAS. Plotted for each of the four motifs is the difference in mean tail length observed for mRNAs of the N37-PAS-N17 library containing that motif at each position along the 3′ UTR. The shaded region marks the PAS. The dashed brown line indicates the difference in mean tail length of all mRNAs in the library. Otherwise, this panel is as in (A).
(C) Influence of the number of the CPEs. Plotted is the difference in mean tail length of mRNAs in the N60-PASmos library containing the indicated numbers of CPEs, comparing between 0 and 7 h post-progesterone treatment. Error bars indicate standard error of the difference between means.
(D) Influence of the number of CPE-like motifs. This panel is as (C), but for CPE-like motifs, UUUUU, UGUUU, GUUUU, UUUGU, and AUUUU, which were the top five motifs associated with tail lengthening, after excluding all variants containing UUUUA.
(E) Effect of CPE flanking nucleotides. Plotted is the difference in mean tail length observed for mRNAs in the N60-PASmos library containing one CPE and the indicated nucleotide at the indicated position, comparing between 0 and 7 h post-progesterone treatment. Shaded areas along the lines indicate standard error of the difference between means.
(F) Influence of the number of PASs. Plotted is the difference in mean tail length of mRNAs in the CPEmos-N60 library containing the indicated numbers of PASs, comparing between 0 and 5 h post-progesterone treatment; otherwise, as in (C).
(G) Effect of the distance between the PAS and the mRNA 3′ end. Plotted is the difference in mean tail length of mRNAs in the CPEmos-N60 library containing the indicated PAS element at each position along the variable region of the 3′ UTR; otherwise as in (A).
(H) Effect of PAS flanking nucleotides. Plotted is the difference in mean tail length observed for mRNAs in the CPEmos-N60 library containing one PAS (AAUAAA) and the indicated nucleotide at the indicated position relative to the PAS, comparing between 0 and 5 h post-progesterone treatment; otherwise, as in (E).
(I) Association of 3′ UTR sequence features with tail-length changes of endogenous mRNAs of frog oocytes. The heatmaps on the left compare median tail lengths of frog oocyte mRNAs collected at the indicated time after progesterone treatment with those in oocytes not treated with progesterone, with each row representing a unique 3′ UTR of an mRNA with a defined poly(A) site. Also indicted for each of these UTRs is the CPE-PAS distance and the minimal and maximal tail-length change over the 7 h treatment. UTRs are grouped based on the presence of a canonical PAS (within 150 nt of the 3′ end) and the number of CPEs (within 1,000 nt of the 3′ end). Only UTRs with poly(A) sites that had more than 50 poly(A) tags in all datasets were analyzed. For UTRs that contained more than one PAS, the one closest to the 3′ end was used. For UTRs that contained more than one CPE, the one closest to the PAS was used to calculate the CPE-PAS distance.
See also Figure S2.
Figure 4. Early fish embryos use a more permissive CPE and undergo more global polyadenylation
(A) Experimental scheme for mRNA library injection and sample collection.
(B) Effects of 5-mers on tail lengths of mRNAs in the N37-PAS-N17 library during zebrafish early embryogenesis. Plotted for each 5-mer are the mean tail lengths of mRNAs containing that 5-mer within their 3′ UTRs. The inset shows the schematic of the injected N37-PAS-N17 library.
(C) Mean tail-length changes associated with each 8-mer in the 3′ UTRs of mRNAs of the N37-PAS-N17 library, plotted as in Figure 3B.
(D) Effects of the CPE and related motifs on poly(A)-tail length in different biological contexts. Shown are mean tail-length changes of mRNAs in the N37-PAS-N17 library containing the indicated sequence motifs (plotting changes relative to mean tail lengths of all variants; V represents A, C, or G; S represents C or G), comparing between 0 and 7 h post-progesterone treatment in frog oocytes, between 0.1 and 1 hpi in frog embryos, and between 0 and 1 hpi in zebrafish embryos (right). Error bars indicate standard error of the difference between means.
(E) Preferential tail lengthening of CPE-containing mRNAs. Plotted are cumulative distributions of tail-length changes for endogenous zebrafish mRNAs with 3′ UTRs that either contained or did not contain UUUUW, comparing between 1-cell and high-stage embryos. The numbers of unique mRNAs in each group are in parentheses; p value, Mann-Whitney U test.
See also Figure S4.
Figure 5. Translation in frog oocytes and early embryos is controlled by both tail-length-dependent and tail-length-independent mechanisms
(A) Experimental scheme for translational profiling of mRNAs in frog oocytes by ribosome pelleting. For each mRNA, TE is measured as the fold enrichment observed between the pellet and the input.
(B) Effects of 6-mers on the TE of mRNAs in the N60-PASmos library during frog oocyte maturation. Results are shown for control prophase I-arrested oocytes that had been treated with ethanol (left) and for maturing oocytes that had been treated with progesterone (right).
(C) TE and tail lengths associated with each 6-mer in the 3′ UTRs of the N60-PASmos library. Shown for each 6-mer is the mean TE observed for mRNAs with that 6-mer, plotted as a function of mean tail length for prophase I-arrested oocytes (7 h post-ethanol treatment, left) and matured oocytes (7 h post-progesterone treatment, right). Colored points indicate 6-mers containing either a CPE (red) or C-rich element (blue).
(D) Tail-length-dependent and -independent translational regulation of CPE-containing mRNAs in frog oocytes. Shown at the top for the CPE and a related motif are TEs of mRNAs in the N60-PASmos library that contained the indicated number of motifs. Shown at the bottom are mean tail lengths of mRNAs in the N60-PASmos library that contained the indicated number of motifs. Increased tail length and translation associated with UUUUU were presumably from UUUUU motifs that preceded an A. Results are shown for prophase I-arrested oocytes (7 h post-ethanol treatment, left) and matured oocytes (7 h post-progesterone treatment, right); ∗adjusted p < 0.05, one-sided binomial test. Error bars indicate standard error.
(E) Translational repression by C-rich elements and the CPE in frog oocytes. On the left are schematics of reporters that examined the effects of elements in two different 3′ UTR sequence contexts. On the right are NanoLuc signals for each reporter after normalization to a co-injected firefly luciferase reporter. Error bars indicate the standard deviation of biological triplicates; p values are from Student’s t tests between indicated groups; WT, wild type.
(F) Translation and tail-length regulation by 3′ UTR sequence in frog embryos. This panel is as in (C), but for embryos at stages 6 (left) and 12 (right), and 6-mers containing either a miR-427 site (green) or an ARE (purple) are also colored.
See also Figure S5.
Figure 6. Tail-length control is conserved in mouse and human oocytes
(A) Poly(A) tail-length change during mouse oocyte maturation. Shown is the plot comparing median tail lengths in mouse MII oocytes to those in mouse GV oocytes. Results are shown for mouse cytoplasmic mRNAs (gray), zebrafish spike-in mRNAs (blue), and poly(A) standards (red). Numbers of mRNAs are indicated in parentheses. Only RNAs with ≥50 tags in each sample were analyzed. On the sides are density distributions for each sample.
(B) Coupling of TE with tail length in mouse oocytes. Shown is the relationship between differences in TE and differences in poly(A)-tail length for mouse oocyte mRNAs, comparing values measured in GV oocytes with those measured in MII oocytes.
(C) ROC curves testing the ability of the UUUUA and previously proposed CPEs to classify endogenous mRNAs as subject to cytoplasmic polyadenylation during mouse oocyte maturation; otherwise, as in Figure 1F.
(D) As in (B), but for human oocytes.
(E) As in (C), but for human oocytes.
(F) Effect of 3′ UTR sequence features on tail-length changes of mRNAs as mouse GV oocytes mature to MII oocytes; otherwise, as in Figure 2I.
(G) As in (E), but for human oocytes.
(H) Genes with substantial mRNA tail lengthening (≥15 nt) in human (H.s.), mouse (M.m.), and frog (X.l.) oocytes. Heatmaps show tail-length changes (left) and TE changes (right), comparing between human GV and MII oocytes, mouse GV and MII oocytes, and frog oocytes 0 and 7 h post-progesterone treatment. Gray indicates values not available. Asterisks indicate genes with ≥2-fold TE changes.
See also Figure S6.
