mRNA 3′ uridylation and poly(A) tail length sculpt the mammalian maternal transcriptome

Abstract

A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome1,2. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3′ terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3′ oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3′ terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.

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Figure 1: The oocyte transcriptome is characterized by short poly(A) tails and oligo-uridylation.
Figure 2: Infertility and defective oocyte maturation in Tut4/7cKO mice.
Figure 3: Short poly(A) tail length and TUT4/7-mediated oligo-uridylation target oocyte transcripts for degradation.
Figure 4: TUT4/7-dependent RNA uridylation in different tissues and cell lines does not impact gene expression.

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Acknowledgements

The research leading to these results has received funding from the Wellcome Trust (Award 106144). This study was technically supported by EMBL Monterotondo’s genome engineering and microscopy core facilities.

Author information

M.M. and C.M. contributed to the design, execution and analysis of most experiments. M.M. performed most of the molecular biology for library generation and much of the bioinformatics analyses. C.M. performed all inducible conditional genetics in somatic cells and tissues. M.D.G., C.A. and I.I. performed oocyte analysis and collection. D.M.V. performed the small RNA sequencing analysis. P.N.M. performed the in vitro embryo development experiments. J.P. and V.B. optimized gene expression analysis from oocytes. J.P., P.C. and V.B. optimized spike-ins and sequencing of TAIL-seq libraries. A.J.E. constructed the TAIL-seq pipeline to process the raw sequencing data and oversaw all bioinformatic analyses performed. D.O’C. conceived and supervised this study and wrote the final version of the manuscript.

Correspondence to Dónal O’Carroll.

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Extended data figures and tables

Extended Data Figure 1 Generation and analysis of Tut4 and Tut7 reporter alleles.

a, Tut4HA-GFP reporter allele targeting strategy. Depicted at the top is the domain organization of the TUT4 protein consisting of C2H2 and CCHC-type zinc-finger domains, an inactive nucleotidyl-transferase (iNT) domain, a polymerase-associated domain (PAD) and a nucleotidyl-transferase (NT) domain. Below this, the 5′ portion of the Tut4 locus, the targeting vector for the insertion of Flag-HA2-PreScission-His6x-eGFP (FHpHG) into the first coding exon of Tut4, and the targeted Tut4 locus before and after Cre-mediated deletion of the loxP site (indicated by red triangles)-flanked neomycin (neo) resistance cassette are shown. The position of the diphtheria toxin A (DTA) selection marker within the targeting vector is shown. The NheI restriction sites as well as the expected Southern fragments detected by the 3′ probe are indicated. Depicted at the bottom is the structure of the resulting HA–GFP–TUT4 protein. b, Tut7HA-GFP reporter allele targeting strategy for the insertion of Flag-HA2-eGFP (FHG) into the first coding exon of Tut7 is depicted as in a. The BamHI restriction sites as well as the expected Southern fragments detected by the 3′ probe are indicated. c, Southern blot of NheI-digested genomic DNA from wild-type, Tut4HA-GFP-Neo/+ and Tut4HA-GFP/+ tails hybridized with the 3′ probe shown in a. d, Southern blot of BamHI-digested genomic DNA from wild-type, Tut7HA-GFP-Neo/+ and Tut7HA-GFP/+ tails hybridized with the 3′ probe shown in b. e, Western blot using anti-HA, anti-TUT4 and anti-tubulin antibodies on testis whole-cell extracts of wild-type, Tut4−/− and Tut4HA-GFP/HA-GFP mice is shown. f, Western blot using anti-HA, anti-TUT7 and anti-tubulin antibodies on testis whole-cell extracts of wild type, Tut7−/− and Tut7HA-GFP/HA-GFP mice is shown. g, Confocal immunofluorescence micrographs of wild-type, Tut4HA-GFP/HA-GFP and Tut7HA-GFP/HA-GFP ovary sections stained with anti-HA antibody (green) and Hoechst 33342 (blue) are shown as indicated. Dashed white circles highlight the oocyte within the follicle. Insets are magnifications of the oocytes. Scale bars, 30 μm.

Extended Data Figure 2 Analysis of DNA spike-in standards used for TAIL-seq library preparations and mRNA uridylation counts.

a, Cumulative frequency of poly(A) length determined by the TAIL-seq algorithm for the different standards used. b, Relative read recoveries of the spike-ins according to their poly(A) tail length. All standards were normalized to the spike-in of 0 length. The linear regression line is shown in blue. a indicates slope; b indicates the y-axis intercept. c, Mono- and oligo-uridylation counts for transcript with short poly(A) tail length (≤30 nucleotides). The number of transcripts with short poly(A) tail length, and the number of genes represented by these transcripts are shown for the indicated tissues and cell lines.

Extended Data Figure 3 Generation of conditional, null and catalytically inactive alleles for Tut4 and Tut7.

a, Tut4 targeting and deletion strategy. Domain organization of TUT4 and TUT7 is presented as in Extended Data Fig. 1a. A portion of the Tut4 locus, the targeting vector used to introduce the loxP sites into the Tut4 locus to flank exons 14 and 15, and the targeted Tut4 gene before and after Flp-mediated recombination as well as after Cre-mediated recombination are shown. Green and red triangles represent FRT and loxP sites, respectively. Rectangles indicate the position of the neomycin and diphtheria toxin A selection marker genes. The EcoRV and BamHI restriction sites as well as the expected Southern fragments detected by the 5′ probe are indicated. b, Tut7 targeting and deletion strategy. A portion of the Tut7 locus, the targeting vector used to introduce the loxP sites into the Tut7 locus to flank exon 15, and the targeted Tut7 gene before and after Flp-mediated recombination as well as after Cre-mediated recombination are shown. The EcoRV restriction sites as well as the expected Southern fragments detected by the 5′ probe are indicated. c, Southern blot of BamHI-digested genomic DNA from wild-type and Tut4Neo/+ tails hybridized with the 5′ probe shown in a. d, Southern blot of EcoRV-digested genomic DNA from wild-type, Tut7 fl/fl and Tut7−/− tails hybridized with the 5′ probe shown in b. e, Western blot using anti-TUT4, anti-TUT7 and anti-tubulin antibodies on testis (Tes) and thymus (Thy) whole-cell extracts of wild-type, Tut4−/− and Tut7−/− mice. f, Targeting strategy used to generate the Tut4AAD allele. A portion of the Tut4 locus, the targeting vector used for the introduction of the Asp1026Ala and Asp1028Ala mutations and the targeted Tut4 gene before and after Flp-mediated recombination are shown. The BamHI and PmeI restriction sites as well as the expected Southern fragments detected by the 5′ probe are indicated. g, Chromatograms showing a portion of Tut4 exon 15 highlighting the two aspartate and alanine codons of wild-type and Tut4AAD/AAD mice, respectively. h, Southern blot of BamHI-PmeI-digested genomic DNA from wild-type and Tut4AAD/+ tails hybridized with the 5′ probe shown in f. i, Western blots using anti-TUT4 and anti-AGO2 antibodies on testis whole-cell extracts from wild-type and Tut4AAD/AAD mice.

Extended Data Figure 4 Antral follicle morphology and in vitro oocyte maturation after Tut4 and Tut7 deletion.

a, The frequency of developing and developed antral follicles in Tut4/7CTL and Tut4/7cKO mice is shown. N.S., not significant Fisher’s exact test. Tut4/7CTL, n = 52 follicles, n = 5 mice; Tut4/7cKO, n = 49 follicles, n = 5 mice. b, Left, representative confocal immunofluorescence micrographs of Tut4/7CTL and Tut4/7cKO fully grown GV oocytes stained with Hoechst 33342. The non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) states are indicated. Dashed white circles highlight the oocytes. Scale bars, 20 μm. Right, the frequency of non-surrounded nucleolus and surrounded nucleolus states in Tut4/7CTL and Tut4/7cKO GV oocytes. *P < 0.05, Fisher’s exact test. Tut4/7CTL, n = 85 oocytes, n = 3 mice; and Tut4/7cKO, n = 135 oocytes, n = 3 mice. c, In vitro oocyte maturation of Tut4/7CTL, Tut4/7cKO and Tut4/7cAAD oocytes. The frequencies of GV oocytes, germinal vesicle breakdown (GVBD) oocytes and oocytes with polar bodies (PB) are shown at different time points after collection and culture. Tut4/7CTL, n = 160 oocytes, n = 4 mice; Tut4/7cKO, n = 194 oocytes, n = 3 mice; and Tut4/7cAAD, n = 183 oocytes, n = 3 mice. d, Representative confocal immunofluorescence micrographs of in vitro maturating Tut4/7CTL, Tut4/7cKO and Tut4/7cAAD oocytes stained with anti-tubulin antibody (green), and DNA stained with Hoechst 33342 (red) and the indicated time points are presented. Scale bars, 20 μm. Source data

Extended Data Figure 5 Validation of gene expression changes upon loss of TUT4 and TUT7 in different tissues and cell lines.

af, Relative expression of genes in Tut4/7CTL and Tut4/7cKO GV oocytes (a, b) as well as Tut4/7CTL and Tut4/7iKO liver (c), bone marrow (d), MEFs (e) and ES cells (f) as determined by qRT–PCR is shown. Depicted are representative genes that have been determined to be upregulated (a) and unchanged (bf) according to microarray profiling. Ct values were normalized against levels of Polr2a, Gapdh and Sod1. Data are mean and range of biological replicates (n = 3). The fold change is indicated. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed Student’s t-test. Source data

Extended Data Figure 6 RNA profiling analysis of Tut4/7cKO and Tut4/7cAAD GV oocytes.

a, b, Expression scatterplot showing relative average expression of transcripts between Tut4/7CTL and Tut4/7cAAD (a) as well as Tut4/7cKO and Tut4/7cAAD (b) GV oocytes. Significantly deregulated (P < 0.01, moderated t-statistic) genes with a fold change greater than 2.0 are shown in red. c, Gene Ontology analysis for upregulated genes in Tut4/7cKO and Tut4/7cAAD GV oocytes; the top ten most significant pathways identified are shown. d, Left, diagram of oocyte growth stages that have been analysed. Right, the top 30 clusters resulting from the clustering analysis according to gene expression patterns across oocyte growth stages (x axis) are shown. The average expression change for genes within the cluster is indicated by a black line, with the s.d. range indicated in grey.

Extended Data Figure 7 Loss of TUT4 and TUT7 reduces both mono- and oligo-uridylation in different tissues and cell lines.

a, Transcript count for upregulated and not upregulated genes in Tut4/7CTL and Tut4/7cKO GV oocytes. The number of transcripts and the number of genes represented by those transcripts (in parentheses) are presented. P values determined using the Pearson’s chi-squared test. bd, Quantification of mRNA 3′ terminal mono-uridylation (b), oligo-uridylation (c) and total uridylation (d) of all transcripts (left) as well as those with short (≤30 nucleotides) (centre), and long (>30 nucleotides) (right) poly(A) tails of Tut4/7CTL and Tut4/7cKO GV oocytes as well as Tut4/7CTL and Tut4/7iKO liver, bone marrow, MEFs and ES cells. Data are mean and range of biological replicates. The fold reduction in uridylation is indicated. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. e, Southern blots of EcoRV-digested genomic DNA from Tut4/7CTL and Tut4/7iKO liver and bone marrow confirming deletion of Tut4 and Tut7 after tamoxifen administration. The expected sizes for the wild-type, the conditional (flox) and the null alleles are indicated. Source data

Extended Data Figure 8 Deletion of Tut4 and Tut7 in mouse embryonic fibroblasts and stem cells.

a, Strategy and timing for the acute deletion of Tut4 and Tut7 in MEFs. TMX, 4-hydoxytamoxifen. b, Southern blots of EcoRV-digested genomic DNA from Tut4/7CTL and Tut4/7iKO MEF cell lines confirming deletion of Tut4 and Tut7. The expected sizes for the wild-type, conditional (flox) and null alleles are indicated. c, Western blots probed with the indicated antibodies from Tut4/7CTL and Tut4/7iKO MEF whole-cell extracts. d, Growth curve of two independent Tut4/7CTL and Tut4/7iKO MEF cell lines. The mean and s.d. of three technical replicates are shown. e, Strategy and timing for the acute deletion of Tut4 and Tut7 in mouse ES cells. f, Southern blots of EcoRV-digested genomic DNA from Tut4+/fl;Tut7+/fl;R26ERT-cre/+ and Tut4 fl/fl;Tut7 fl/fl;R26ERT-cre/+ ES cell lines before (TMX−) and after (TMX+) deletion of Tut4 and Tut7. The expected sizes for the wild-type, conditional (flox) and null alleles are indicated. g, Western blots probed with the indicated antibodies from Tut4/7CTL and Tut4/7iKO ES cell whole-cell extracts. h, Representative images showing morphology of Tut4+/fl;Tut7+/fl;R26ERT-cre/+ and Tut4 fl/fl;Tut7 fl/fl;R26ERT-cre/+ ES cell lines before (TMX−) and after (TMX+) deletion of Tut4 and Tut7. Scale bar, 200 μm. i, Growth curve of Tut4+/fl;Tut7+/fl;R26ERT-cre/+ and Tut4 fl/fl;Tut7 fl/fl; R26ERT-cre/+ES cell lines before (TMX−) and after (TMX+) deletion of Tut4 and Tut7. The growth of each line was measured in triplicates. The mean and s.d. of three independently derived cell lines are shown. j, Alkaline phosphatase staining of untreated (TMX−) and tamoxifen-treated (TMX+) Tut4+/fl;Tut7+/fl;R26ERT-cre/+ and Tut4 fl/fl;Tut7 fl/fl;R26ERT-cre/+ ES cell lines. k, Relative expression of pluripotency markers in Tut4/7CTL and Tut4/7iKO ES cell lines as determined by qRT–PCR from three biological replicates. Data are mean and range. The fold change is indicated. l, Representative images showing morphology of Tut4/7CTL and Tut4/7iKO embryoid bodies. Magnification, 20×. m, Representative images showing morphology of neural progenitors derived from of Tut4/7CTL and Tut4/7iKO ES cells. Scale bar, 100 μm. n, Relative expression as determined by qRT–PCR of endoderm and mesoderm markers in embryoid bodies derived from Tut4/7CTL and Tut4/7iKO ES cells. Data are mean and range from three biological replicates. The fold change is indicated. o, Expression as determined by qRT–PCR presented as fold change of pluripotency and neural progenitor markers after differentiation of Tut4/7CTL and Tut4/7iKO ES cells into neural progenitors. The fold change is indicated. Data are mean and range. *P < 0.05, two-tailed Student’s t-test. p, Frequency of Tut4/7CTL and Tut4/7iKO embryoid bodies with spontaneous contractile activity. Data are mean and range from three biological replicates, each determined from 48 embryoid bodies. The fold change is indicated. Source data

Extended Data Figure 9 3′ mRNA terminal transcriptome cytidylation and guanylation are not affected after Tut4 and Tut7 deletion.

a, b, Quantification of mRNA 3′ terminal cytidylation (a) and guanylation (b) of all transcripts (left), transcripts with short poly(A) tails (≤30 nucleotides) (centre) and long poly(A) tails (>30 nucleotides) (right panel) from Tut4/7CTL and Tut4/7cKO GV oocyte or Tut4/7iKO liver, bone marrow, MEF and ES cell transcriptomes. The fold change in modification levels is indicated. *P < 0.05, two-tailed Student’s t-test. Data are mean and range from biological replicates. Source data

Extended Data Figure 10 Loss of TUT4 and TUT7 specifically reduces terminal miRNA uridylation levels and modestly impacts miRNA expression levels.

a, Quantification of the relative abundance of let-7 family members, the five most abundant miRNAs, and all other miRNAs in Tut4/7CTL and Tut4/7iKO liver, bone marrow, MEFs and ES cells. The fold change in relative let-7 expression is depicted. be, Quantification of 3′ uridylation (b), adenylation (c), cytidylation (d) and guanylation (e) frequency of all miRNAs and let-7 family members in Tut4/7CTL and Tut4/7iKO cell lines and tissues. Modifications involving one or more than one nucleotide are distinguished. Data are mean and range of biological replicates. The fold change in modification frequency between Tut4/7CTL and Tut4/7iKO is indicated. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. Source data

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Morgan, M., Much, C., DiGiacomo, M. et al. mRNA 3′ uridylation and poly(A) tail length sculpt the mammalian maternal transcriptome. Nature 548, 347–351 (2017) doi:10.1038/nature23318

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