January 1, 2018;
Terminal Uridylyltransferases Execute Programmed Clearance of Maternal Transcriptome in Vertebrate Embryos.
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3'' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4
and TUT7 (TUT4
/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4
/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.
gastrulation with mouth forming second
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Figure 2. (A) Average 3′ non-A nucleotide addition per tail, determined using all the mRNA reads containing 5–15 nt poly(A) tails. The light brown shadow roughly spans the MZT duration. X. laevis stages are indicated in the Nieuwkoop and Faber (NF) stage numbers.
(B) Poly(A) length and uridylation status of two representative genes. A dot represents a single sequence read. The color indicates the length of U at the 3′ end. The number of dots in any sample is proportional to the relative RNA abundance measured by RNA-seq.
Figure 3. (A and B) Average uridylation count per tail, measured after the injection of control or TUT4/7 translation-blocking morpholino (MO) into zebrafish (A) or frog (B) embryos. y axis shows the arithmetic mean of gene-level average U counts in the short poly(A) tails (5–15 nt). In zebrafish experiments, all SEM of the average U counts were smaller than 0.012. SEM is shown as vertical bars in (B).
(C) Average uridylation count per tail (5–15 nt) in 4 hpf zebrafish embryos injected with control or TUT4/7 MOs. Each dot represents a gene with ≥10 short poly(A) tags in both samples. The color of a dot shows the density of dots nearby; blue (low), green-yellow (medium), or red (high).
(D) Ribosome density distribution of expressed genes in the 4 hpf zebrafish embryo. The curves show all non-histone coding genes with ≥20 reads by RNA-seq after trimmed mean of M-values (TMM) normalization. Genes tut7 and dis3l2 are indicated by red and blue vertical bars in both full (lower left) and zoomed-in (upper right) planes.
Figure 4. (A and B) Zebrafish (A) and frog (B) embryos showing the effects of TUT4/7 morpholinos (MOs). In (B), black arrows indicate the edges of the blastopore (gastrula) and the positions of neural tube closure (neurula).
(C) Zebrafish embryos displaying the rescue effects after co-injection of TUT7 morpholino and tut7 mRNA. (top) Representative images of embryos injected with each treatment as annotated above. The numbers on the bottom right corner of each panel indicate the embryo count showing the same morphological characteristics as the presented image and the total number of embryos used for the experiment, respectively. (bottom) The number of embryos that progressed to the beginning of epiboly. An asterisk indicates a significant effect (p <1.17 × 10−5; Fisher’s exact test).
(D) Frog embryos demonstrating the rescue effect of the tut7 mRNA injection. (top) The embryos were injected with control or TUT4/7 morpholino, along with GFP mRNA as a control (leftmost and the second), frog wild-type tut7 mRNA (third), or frog tut7 mRNA with a “DADA” mutation in its active site (rightmost). Black arrows indicate the positions of the blastopore. Numbers on the bottom right corner of each panel indicate the embryo counts with the shown morphology and total counts, respectively. (bottom) The number of embryos without defects in blastopore closure. An asterisk indicates a significant effect (p <1.15 × 10−3; Fisher’s exact test; two conditions resulted in the exactly same counts by chance).
(E) Average length of U tails per tail of all 5–15 nt poly(A) tails in frog stages 9 and 12.