The catalytic activity of microRNA Argonautes plays a modest role in microRNA star strand destabilization in C. elegans

Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use genome editing in C. elegans to examine the role of miRNA-guided slicing in organismal development. In contrast to previous work, slicing-inactivating mutations did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the absence of slicing, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on slicing for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on slicing. Gene expression changes were consistent with negligible to moderate loss of function for miRNA guides whose star strand was upregulated, suggesting a reduced proportion of mature miRISC in slicing mutants. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, a factor in the Target-Directed miRNA Degradation (TDMD) pathway. Overall, this work defines a role for miRNA Argonaute slicing in star strand decay; future work should examine whether this role could have contributed to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.


Figure S1 .
Figure S1.Western blot of FLAG-tagged wild type and AEDH mutant Argonautes Figure S2.Effect of RNAi on phenotype in AEDH mutant backgrounds Figure S3.Spike in normalization supports minor changes in miRNA abundances Figure S4.Validation of differential expression by qPCR Figure S5.Standard curves for absolute miRNA quantification by qPCR Figure S6.Polymorphism in gpap-1 5' UTR likely contributes to decreased expression Figure S7.Reproducibility of miRNA changes in ebax-1(null) and across two alg-1(AEDH); alg-2(AEDH) experiments Figure S1.Western blot of FLAG-tagged wild type and AEDH mutant Argonautes.Two to three biological replicates of each genotype were quantified.For experimental samples, 25µg of protein is loaded per lane.A dilution series of a mixed-stage sample is included to calibrate fold changes.For experimental samples, FLAG signal was divided by loading control signal in the same lane to determine normalized FLAG signal; for dilution series samples, normalized FLAG signal was calculated by dividing each FLAG signal by the average of the wild type experimental sample loading control signals.Each normalized FLAG signal was then divided by the average normalized FLAG signal for all wild type samples.

Figure S2 .
Figure S2.Effect of RNAi on phenotype in AEDH mutant backgrounds.RNAi and phenotype scoring was performed as in Bouasker, et al.Eight plates were scored per genotype/RNAi and treated as replicates.n > 470 animals per genotype/RNAi condition.Mean and SEM are plotted.AEDH mutations do not behave as loss-of-function alleles since they display no synthetic lethality with RNAi of the other ALG paralog.

Figure S3 .
Figure S3.Spike in normalization supports minor changes in miRNA abundances.A) MA plot showing average abundance on X-axis and log2(fold change) on Y-axis.miRNAs showing significant changes in alg-1(AEDH); alg-2(AEDH) versus wild type are shown in blue (miRNA guide strands) or pink (star strands) (DEseq2 FDR<0.1).Reads were normalized to exogenous spike-ins (See methods and Table S2).Three (L4) or four (embryo and young adult) biological replicates of each genotype were analyzed.(B) MA plot as in (A), with all guide strands shown in black and all star strands in pink.(C) Summary of log2(fold change) values for all guides or star strands in each stage in alg-1(AEDH); alg-2(AEDH) versus wild type.Log2(fold change) of star strands were compared to those of guide strands in the same sample by one-way ANOVA followed by Sidak's multiple comparisons test.***p<0.0001

Figure S5 .
Figure S5.Standard curves for absolute quantification by qPCR.Synthetic RNA oligonucleotides were diluted to indicated concentrations, and 1.66µl was used in 5µl RT reactions and downstream miRNA-Taqman as indicated in methods.Average of three technical replicates is shown.Highest and lowest concentrations were excluded from the curve if they were outside the linear range.All experimental sample concentrations fell within the linear range of the respective assay.

Figure S6 .
Figure S6.Polymorphism in gpap-1 5' UTR likely contributes to decreased expression.A) Left: Absolute quantification by qPCR of mir-63 and mir-72 guide and star strands Right: Strand bias calculated from absolute quantification by qPCR.Mean and SEM of three to four biological replicates per condition are shown.B) One gene was identified as mis-regulated across more than one developmental stage in the alg-1(AEDH);alg-2(AEDH) mutant strain.Close examination of RNA-seq data revealed that this gene, gpap-1, contains a polymorphism in its 5' UTR in the mutant strain.

Figure S7 .
Figure S7.Reproducibility of miRNA changes in ebax-1(null) and across two alg-1(AEDH); alg-2(AEDH) experiments.A) MA plots showing average abundance on X-axis and log2(fold change) on Y-axis.miRNAs showing significant changes are highlighted in light blue or purple (DEseq2 FDR<0.1);upregulated miRNAs that were previously noted in Shi, et al are in purple.Four biological replicates of each genotype were analyzed.B) Correlation of changes induced by alg-1(AEDH); alg-2(AEDH) in wild type experiment 1 (X-axis) or experiment 2 (Y-axis).Each experiment consists of four biological replicates of each genotype.Only miRNAs with baseMean > 50 in wild type experiment 1 are included.(A-B) All samples are from young adults.