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. 2021 Nov 8;49(19):11167-11180.
doi: 10.1093/nar/gkab840.

Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans

Karl-Frédéric Vieux  1 , Katherine P Prothro  1   2 , Leanne H Kelley  3 , Cameron Palmer  1 , Eleanor M Maine  3 , Isana Veksler-Lublinsky  4 , Katherine McJunkin  1
Affiliations

Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans

Karl-Frédéric Vieux et al. Nucleic Acids Res. .

Abstract

microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2-here named GLD-2-related 2 (GLDR-2)-is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

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Figures

Figure 1.
Figure 1.
Characterization of miRNA tailing in C. elegans. (A) 2D matrix representing the truncation and tailing status of the indicated miRNAs. The sizes of the dots represent the proportion of reads, with the canonical sequence at zero on both axes, and increasingly trimmed or tailed species plotted to the left on the x-axis or upward on the y-axis, respectively. Bottom: Number of reads of most abundant mir-70-3p species in one wild type adult library, with the canonical sequence being the most abundant. (B) Mean and standard deviation of prevalence of single nucleotide 3′ terminal additions of each indicated nucleotide from three biological replicates. (C) Schematic of miRNA tailing preceding or following Dicer-mediated cleavage of the miRNA precursor. (D) Comparison of prevalence of mono-adenylation and mono-uridylation on 3p- versus 5p-derived miRNAs. One biological replicate is shown. (E) Meta-analysis of three published datasets shows tailing across C. elegans developmental stages. Prevalence of mono-uridylation of miRNAs across development is shown. Only miRNAs with >50 RPM in the indicated library were analyzed. (B, D) Only miRNAs with >50 RPM in all biological replicates were analyzed. (B, D, E) Each dot represents an individual miRNA.
Figure 2.
Figure 2.
miRNA uridylation requires CID-1. (A) Heatmap summarizing percent of mono-uridylation across each miRNA (rows) in each RNAi condition (columns). All miRNAs with >50 RPM in all empty vector replicates and an average of > 1% mono-uridylation across the three empty vector replicates are shown. Arrowheads above heatmap indicate columns significantly different than vector (cid-1 and exos-4 RNAi). ***P-value = 0.0003, *P-value = 0.016. Open circle indicates pup-2 RNAi, which does not have an effect. (B) Percent mono-uridylation in vector or cid-1 RNAi. Each column is an individual miRNA. All miRNAs with > 50 RPM in all empty vector replicates are shown. Three biological replicates of empty vector are shown in gray. (C) Phylogenetic relationship of CID-1, PUP-2, TUT4 and TUT7. (Note the following alternative names for these genes CID-1/CDE-1/PUP-1, PUP-2, TUT4/TENT3A/ZCCHC11/PAPD3 and TUT7/TENT3B/ZCCHC6/PAPD6). (D) Percent mono-uridylation in indicated genotype. All miRNAs with >50 RPM in all wild type replicates are shown. Mean and standard error are shown for three biological replicates per genotype.
Figure 3.
Figure 3.
miRNA adenylation requires F31C3.2/GLDR-2. (A) Heatmap summarizing percent of mono-adenylation across each miRNA (rows) in each RNAi condition (columns). Arrowhead above heatmap indicates column significantly different from vector, corresponding to F31C3.2 RNAi, which reduces adenylation. ***P-value = 0.0003 (B) Percent mono-adenylation in vector or F31C3.2/GLDR-2 RNAi. Three biological replicates of empty vector are shown in gray. (A, B) All miRNAs with >50 RPM in all empty vector replicates and an average of >1% mono-adenylation across the three empty vector replicates are shown. (C) Phylogenetic tree of F31C3.2/GLDR-2, GLD-2 and WISP. (Note that GLD2 in humans is also known as TENT2/TUT2/PAPD4/APD4). (D) Percent mono-adenylation in indicated genotypes/RNAi. Mean and standard error for four biological replicates are shown. MiRNAs shown are the same as those in (A, B).
Figure 4.
Figure 4.
Characterization of miRNA half-lives in C. elegans. (A) Schematic of time courses after pash-1 inactivation used for measuring miRNA decay. (B) Log2 fold change of miRNA reads compared to 0 h after PASH-1 inactivation (y-axis) versus abundance at 0 h (x-axis). Reads were normalized to spike-ins. Mean and standard error of two biological replicates are shown. (C) Representative time course data plotted as fold change, with exponential decay model and 95% prediction bands (dashed lines). Data for two biological replicates is shown. (D) Distribution of miRNA half-lives modeled from pash-1(ts) time course data. (B–D) Only miRNAs with >50 RPM in both replicates at the 0 h timepoint were included in the analysis.
Figure 5.
Figure 5.
The proportion of tailed and trimmed miRNAs increases over time after biogenesis. (A) Average percent of reads bearing each indicated single-nucleotide tail. (B) Prevalence of mono-uridylation of miRNAs across time course after PASH-1 inactivation in one biological replicate. Each dot represents an individual miRNA. (A, B) One-way ANOVA was used to compare all time points to 0 h; when ANOVA was significant, Dunnett's multiple comparison test was performed, and P-values are shown above graph. (C) Representative plots for indicated miRNAs, where y-axis indicates percent mono-uridylation, and size of dot represents read number. Note that reads here are reads per million spike-in reads, an arbitrary unit.
Figure 6.
Figure 6.
Uridylation does not globally regulate miRNA abundance or decay. (A) Abundance of miRNAs in cid-1 RNAi versus empty vector. Mean and standard error are plotted. (B, C) Abundance of miRNAs in indicated mutant versus wild type. (A–C) Except for cid-1 RNAi (two biological replicates), mean and standard error of three biological replicates are plotted. All miRNAs > 50 RPM in all empty vector or wild type replicates, respectively, are shown. (D) Log2fold change in reads per million from zero to 24 h after PASH-1 inactivation. miRNAs are divided into three plots by the average percent mono-uridylation in empty vector at 0 h. All miRNAs with >50 RPM at 0 h in both empty vector replicates are shown, except for mir-35–41. (E) Log2fold change from zero to 24 h after PASH-1 inactivation for mir-35–41 guide (3p) strands and star (5p) strands. All miRNAs from this group are shown, regardless of abundance, except for mir-35-5p and mir-37-5p which had ∼0 reads in all samples. (F) Abundance of mir-35–41 guide and star strands at 0 h (prior to PASH-1 inactivation). (G) Plots showing percent mono-uridylation and miRNA abundance at zero and 24 h after PASH-1 inactivation. Size of dot represents abundance, and y-axis is percent mono-uridylation.
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References

    1. Dexheimer P.J., Cochella L.. MicroRNAs: from mechanism to organism. Front. Cell Dev. Biol. 2020; 8:409. - PMC - PubMed
    1. Alberti C., Cochella L.. A framework for understanding the roles of miRNAs in animal development. Dev. 2017; 144:2548–2559. - PubMed
    1. Ha M., Kim V.N.. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014; 15:509–524. - PubMed
    1. Bail S., Swerdel M., Liu H., Jiao X., Goff L.A., Hart R.P., Kiledjian M.. Differential regulation of microRNA stability. RNA. 2010; 16:1032–1039. - PMC - PubMed
    1. Rissland O.S., Hong S.-J., Bartel D.P.. MicroRNA destabilization enables dynamic regulation of the miR-16 family in response to cell-cycle changes. Mol. Cell. 2011; 43:993–1004. - PMC - PubMed

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