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. 2022 Aug 9;40(6):111154.
doi: 10.1016/j.celrep.2022.111154.

The developmentally timed decay of an essential microRNA family is seed-sequence dependent

Bridget F Donnelly  1 , Bing Yang  2 , Acadia L Grimme  1 , Karl-Frédéric Vieux  2 , Chen-Yu Liu  2 , Lecong Zhou  2 , Katherine McJunkin  3
Affiliations

The developmentally timed decay of an essential microRNA family is seed-sequence dependent

Bridget F Donnelly et al. Cell Rep. .

Abstract

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here, we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3' end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3' end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3' end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed-sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family's target repertoire.

Keywords: C. elegans; CP: Cell biology; CP: Developmental biology; EBAX-1; ZSWIM8; developmental transitions; embryonic development; miRNA; miRNA decay; miRNA degradation; miRNA tailing; miRNA turnover; microRNA; mir-35; mir-35 family; mir-35-41; post-transcriptional regulation; target-directed miRNA degradation; TDMD.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. mir-35 decay is seed-sequence dependent
(A) Sequences of mir-35–42. Seed sequence in purple. (B) Schematic of the mir-35–41 cluster and mir-42 with sequences of mir-35 and variants. (C and D) Absolute quantification of mir-35 and mir-36 guide strands (C) or star strands (D). (E and F) Log2(fold change) from embryo to L1, calculated from deep sequencing for either the mir-35–42 family (E) or all miRNAs >50 RPM in wild type (F). Note that color of bar indicates strain, not necessarily a mutant miRNA; only mir-35 is mutated in the indicated mutant strains. (E) Two-way ANOVA, followed by Dunnett’s multiple comparisons test. ****p value < 0.0001. (F) Small arrows indicate positions of mir-35–41 on ranked x axis, and arrowhead indicates mir-35 and mutant variants. (C–F) Mean and SEM of three biological replicates are shown.
Figure 2.
Figure 2.. mir-35 3′ end mutants do not alter decay
(A) Sequences of mir-35–42 with the identical seed sequences shown in purple (top). Schematic of the mir-35 3′ end mutants (bottom). (B) Absolute quantification of mir-35 and mir-36 in embryos and L1. Mean and SEM of two to three biological replicates. (C) Absolute quantification of star strands of mir-35 and mutant variants in embryos. (D and E) Log2(fold change) from embryo to L1, calculated from normalized deep-sequencing reads for either the mir-35–42 family (D) or all miRNAs with >50 RPM in wild type (E). Note that color of bar indicates strain, not necessarily a mutant miRNA; only mir-35 is mutated in the indicated mutant strains. (D) Two-way ANOVA was performed, followed by Dunnett’s multiple comparisons test. (E) Small arrows indicate positions of mir-35–41 on ranked x axis, and arrowhead indicates mir-35 and mutant variants. (C–E) Mean and SEM of three biological replicates.
Figure 3.
Figure 3.. Changes in tailing and trimming of mir-35 variants do not correlate with changes in decay
(A) Tailing of each miRNA >50 RPM in embryo. (B) Total tailing (sum of all single nucleotide tails) versus abundance (RPM) for all miRNAs >50 RPM in embryo. (C and D) Tailing in the embryo and L1. (E and F) Length distribution (excluding tail) of mir-35 in wild-type embryo and L1 (E) or mir-35 variants in embryo (F). (A–F) Mean and SEM shown. Wild-type and mutant samples have six and three biological replicates, respectively. (C–F) For each nucleotide, one-way ANOVA was performed, followed by Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, ****p < 0.0001. For (D), p values are described in the text and are not on the graph for simplicity.
Figure 4.
Figure 4.. Reintroducing miRNA-target interactions does not restore decay of a seed mutant variant of mir-35
(A) Representative miRNA-target interactions at the egl-1 3′ UTR. (B) Top: pie charts represent the proportion of the mir-35 miRNA and target molecules that are mutated in each strain. Bottom: log2(fold change) from embryo to L1 in the indicated strains, as measured by Taqman qPCR. Mean and SEM of three biological replicates. (C) Model of conventional TDMD. (D and E) Alternative models for regulation of mir-35 family decay, in which the seed sequence is recognized by a complementary RNA (D) or an RNA-binding protein (RBP) (E).
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