Dipeptidyl peptidase DPF-3 is a gatekeeper of microRNA Argonaute compensation in animals

Abstract

MicroRNAs (miRNAs) are essential regulators involved in multiple biological processes. To achieve their gene repression function, they are loaded in miRNA-specific Argonautes to form the miRNA-induced silencing complex (miRISC). Mammals and C. elegans possess more than one paralog of miRNA-specific Argonautes, but the dynamic between them remains unclear. Here, we report the conserved dipeptidyl peptidase DPF-3 as an interactor of the miRNA-specific Argonaute ALG-1 in C. elegans. Knockout of dpf-3 increases ALG-2 levels and miRISC formation in alg-1 loss-of-function animals, thereby compensating for ALG-1 loss and rescuing miRNA-related defects observed. DPF-3 can cleave an ALG-2 N-terminal peptide in vitro but does not appear to rely on this catalytic activity to regulate ALG-2 in vivo. This study uncovers the importance of DPF-3 in the miRNA pathway and provides insights into how multiple miRNA Argonautes contribute to achieving proper miRNA-mediated gene regulation in animals.

Fig. 1. Dpf-3 physically and genetically interacts with the miRNA pathway.

a IP-MS of ALG-1. The x-axis indicates the fold enrichment of the bait proteins and their interacting partners over control (SART-3). Each dot represents a distinct interacting partner. Specific and significant interacting partners are shown. False Discovery Rate (FDR) of 0.05 was used. Exact p-values were calculated by two-tailed Student t-test and are indicated for each interactor on the Y-axis of the volcano plot. N = 4. b Seam cell number in adult WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) worms. Each dot represents a single animal. The number in parenthesis represents the number of animals scored for each genotype. P-value was calculated by two-tailed Mann-Whitney test. n.s. = non-significant. c Top: DIC pictures showing an example of normal alae structure and gapped alae structure (white arrowhead points to the gap in the structure and the bracket shows the length of the gap). Bottom: Percentage of WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) worms displaying an alae gap phenotype at 20 °C. The number in parenthesis represents the number of animals scored for each genotype. P-values were calculated by two-sided Fisher’s exact test. n.s. = non-significant. d Percentage of WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) worms displaying the bursting through the vulva phenotype at 25 °C. Number in parenthesis represents the number of worms scored. Pictures show an example of a normally developed worm versus a burst worm, where the white arrow points at a normal vulva and the white arrowhead points at the gonad sticking out of the vulva. P-values were calculated by two-sided Fisher’s exact test.

Fig. 2. Removal of dpf-3 in alg-1 loss-of-function animals restores normal miRNA regulation of transcripts.

a ΔLog₂ of RPM of miRNA sequencing data where the WT values are subtracted from the dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) values. Each dot represents a single miRNA, where green dots are miRNAs more expressed than WT and red dots are miRNAs less expressed than WT. Black dots represent miRNAs that are within normal expression thresholds (gray dashed lines). The proportion of miRNAs misregulated by at least ±1 ΔLog₂ appear on top of each condition. Each dot is a mean of 3 biological replicates normalized on the total reads. b ΔLog₂ of RPM of miRNA sequencing data where only miRNAs that were up- or downregulated in alg-1(gk214) animals were selected and quantified in specified genetic backgrounds. The proportion of those miRNAs that are within normal expression thresholds (gray dashed lines) appear on top of each condition. c, d RT-qPCR was performed on total RNA extracts of WT, alg-1(gk214) and dpf-3(xe68);alg-1(gk214) worms to measure the relative expression of the let-7 miRNA family members and their targets. Data are presented as mean values of three biological replicates (white dots) ± SD. MiRNA levels were normalized using sn2841, and miRNA target levels were normalized using tba-1. P-values were calculated by two-tailed Student t-test. n.s. = non-significant.

Fig. 3. ALG-2 compensates for the loss of ALG-1 in dpf-3(xe68);alg-1(gk214) animals.

a Representative western blot of ALG-1/2 protein expression in WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) worms. ACTIN acts as a loading control. The numbers on top of the alg-1(gk214) and dpf-3(xe68);alg-1(gk214) bands represent ALG-2 protein signal intensity relative to alg-1(gk214) when normalized on the ACTIN band. N = 4. b Quantification of the signal intensity of the ALG-2 western blot band across four biological replicates, comparing alg-1(gk214) condition to dpf-3(xe68);alg-1(gk214) condition. Data are presented as mean values of four biological replicates (black dots) ± SD. P-value was calculated by two-tailed one sample Student t-test. c let-7 miRISC purification was performed and followed by western blot analysis to measure ALG-1/2 protein association to let-7. The western blot shown is a representative replicate. Input and purifications are separated as they received different exposure time, but were still ran on the same gel. The numbers on top of the alg-1(gk214) and alg-1(gk214); dpf-3(xe68) bands in the let-7 section represent ALG-2 protein signal intensity relative to alg-1(gk214). N = 3. d Quantification of the signal intensity of the ALG-2 western blot band of the let-7 miRISC purifications across three biological replicates, comparing alg-1(gk214) condition to dpf-3(xe68);alg-1(gk214) condition. Data is presented as mean value of three biological replicates (black dots) ± SD. P-value was calculated by two-tailed one sample Student t-test. e alg-2 RNAi was performed on alg-1(gk214) and alg-1(gk214); dpf-3(xe68) L4 animals (5–10 animals per condition per replicate) and the percentage of lethality in the progeny was measured (approx. 100 progeny per condition per replicate). Data are presented as mean values ± SD of three biological replicate. Each dot represents an independent experiment. P-value was calculated by two-way ANOVA. n.s. = non-significant.

Fig. 4. DPF-3 dipeptidase activity is not contributing to ALG-2 modulation.

a Linear regression of the measured signal intensity of the synthetic ALG-2 N-terminal peptide over time after processing by LC-MS/MS. The detected peptide sequences are listed on top of the graph with the black arrow pointing at the cleavage site. The signal intensity was normalized with a CytC peptide. The red line shows signal intensity of cleaved peptide when incubated with catalytic dead DPF-3. Data are presented as mean values of signal intensity for each time point ± SD. The western blot analysis depicts a representative purification of either wild-type or catalytic dead DPF-3 for incubation with ALG-2 peptide. N = 3. b Schematic of ALG-2 N-terminal protein sequence and the result of the proline mutations inserted by CRISPR-Cas9. c Percentage of proline mutant animals displaying gaps in their alae structure at 20 °C. The numbers on top of the bars in the graph represent the number of animals scored for each genotype. P-value was calculated by two-sided Fisher’s exact test. d RT-qPCR was performed on total RNA extracts of WT, alg-1(gk214) and alg-2(qbc102);alg-1(gk214) worms to measure the relative expression of the let-7 miRNA family members. Data are presented as mean values of 3 biological replicates (white dots) ± SD. MiRNA levels were normalized using sn2841. P-values were calculated by two-tailed Student t-test. n.s. = non-significant. e Western blot of ALG-1/2 protein expression in WT, dpf-3(xe68), alg-2(qbc102), alg-1(gk214), dpf-3(xe68);alg-1(gk214) and alg-2(qbc102);alg-1(gk214) animals. ACTIN acts as a loading control. The numbers on top of the alg-1(gk214), alg-1(gk214); dpf-3(xe68) and alg-2(qbc102);alg-1(gk214) bands represent ALG-2 protein signal intensity relative to alg-1(gk214) when normalized on the ACTIN band.

Fig. 5. DPF-3 does not co-immunoprecipitate with ALG-2.

a Schematic of endogenously V5 tagged ALG-2 protein by CRISPR-Cas9. The V5 tag is represented in yellow. Amino acids position are displayed at the bottom. b Representative western blot of V5::ALG-2 immunoprecipitation in v5::alg-2 (- lanes) or dpf-3::flag-ha;v5::alg-2 (+ lanes) background. The asterisk highlights a DPF-3 isoform of lower molecular weight than expected. ACTIN acts as a loading control. N = 2. c Representative western blot of V5::ALG-2 immunoprecipitation in dpf-3::flag-ha;v5::alg-2 (- lanes) or dpf-3::flag-ha;v5::alg-2;alg-1(gk214) (+ lanes) background. The asterisk highlights a DPF-3 isoform of lower molecular weight than expected. ACTIN acts as a loading control. N = 2.

Fig. 6. Absence of DPF-3 in alg-1 loss-of-function animals increases ALG-2 mRNA abundance.

a RT-qPCR performed on total RNA extracts of WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) animals to measure mature ALG-2 mRNA using primers in exon-exon junctions. Data are presented as mean values of 3 biological replicates (white dots) ± SD. ALG-2 mRNA levels were normalized using tba-1. P-values were calculated by two-tailed Student t-test. n.s. = non-significant. b Top: schematic of ALG-2 unspliced mRNA where half-arrows represent roughly the position and orientation of the primers used for qPCR. Bottom: RT-qPCR performed on total RNA extracts of WT, dpf-3(xe68), alg-1(gk214) and dpf-3(xe68);alg-1(gk214) animals to measure ALG-2 precursor mRNA using primers in intronic regions. Data are presented as mean values of 3 biological replicates (white dots) ± SD. ALG-2 pre-mRNA levels were normalized using tba-1. P-values were calculated by two-tailed Student t-test. n.s. = non-significant.

Fig. 7. Model of DPF-3’s regulation on the interplay between miRNA-specific Argonautes.

In wild-type genetic background, DPF-3 favors interaction with ALG-1 and maintains a normal transcription/splicing rate of ALG-2 mRNA. This allows ALG-1 to assume the bulk of the miRNA gene regulatory function, achieving normal target translational inhibition and keeping the animals healthy. In alg-1(gk214) genetic background, ALG-2 pre-mRNA starts to accumulate but DPF-3 limits its processing, which impedes it to compensate for the absence of ALG-1, leading to target mRNAs misregulation and appearance of deleterious phenotypes. In dpf-3(xe68);alg-1(gk214) conditions, DPF-3’s negative regulation of ALG-2 mRNA processing is relieved and allows it to compensate for the lack of ALG-1 by increased protein abundance and increased active miRISC formation. ALG-2 can now maintain proper target translational inhibition and keep the animals healthy again.