The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

Abstract

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the β and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

Keywords: RNA binding proteins; RNA phosphatase; RNAi; double-stranded RNAs; embryogenesis; germline gene regulation; germline small RNAs; mRNA regulation; regulation of triphosphorylated RNA; spermatogenesis.

Figure 1.. PIR-1 is an RNA polyphosphatase.

(A) Alignment of PIR-1 orthologs from C. elegans (C.e.), Drosophila (D.m.), and human (H.s.), asterisk indicates the catalytic cysteine. (B) Terminator exonuclease assays on ppp-RNA substrates with and without pretreatment by WT or C150S recombinant PIR-1. (C) Terminator exonuclease assays on ppp-RNA duplexed with DNA and RNA (schematic of pretreatments, left). (D) Gel-shift assays on single-stranded ppp-RNA and p-RNA substrates using recombinant WT and C150S PIR-1, visualized by 15% native PAGE and SYBR Gold staining. (E) Gel shift assays followed by SYBR Gold staining (left) and western blot (for detection of His-tagged WT and C150S PIR-1, right) on RNA substrates (as indicated). See also Figure S1.

Figure 2.. PIR-1 interacts with the ERI complex.

(A) Western blot analyses on WT (N2) and pir-1::3xflag-rescued young adults showing proteins present in input (lysate), FLAG IP, and post-IP supernatant. The * and ** indicate unspecified bands. (B) Western blot analyses of PIR-1-associated proteins across developmental stages in N2 and PIR-1::3xFLAG (left) and in PIR-1::GFP lysates (as indicated). The * indicates a background signal that co-migrates with tubulin from binding of the secondary antibody to the heavy chain of the anti-Flag antibody. (C) Gel filtration analysis of pir-1::3xflag lysates followed by western blot analyses (as indicated). Arrowheads indicate molecular weights of size standards. See also Figure S2, Table 1 and Table S1.

Figure 3.. PIR-1 is essential for somatic and germline development.

(A) Schematic of the pir-1 locus indicating genetic lesions used. (B) Schematic of a rescuing transgene with GFP exons indicated in green (top panel) and fluorescence micrographs of an L4 germline stained with DAPI and with anti-GFP (bottom panels). (C) DIC images of a pir-1(+/tm3198) heterozygote and an arrested tm3198 homozygote cultured at 20°C for 96 hours with germlines indicated by yellow highlighting (partly concealed by intestine). (D–F) Fluorescence micrographs of WT (N2) and mutant germlines visualized by DAPI in (D–F) and by PGL-1::RFP fluorescence in (E). Distal germline oriented to left. ‘m’, mitotic zone; ‘tz’, transition zone; ‘p’, pachehytene; and ‘sp’, spermatids indicated in (D). Abnormal chromosome bridging is indicated with red arrows in (F). See also Figure S3.

Figure 4.. PIR-1 is required for the biogenesis of 26G-RNAs and non-WAGO-bound 22G-RNAs.

(A) Venn diagrams showing relative abundance of small RNA species in pir-1(tm3198) mutants and control avr3x animals. For each strain, small RNA composition was calculated as the average of two replicas. (B) Western blot analyses of DCR-1 and PRG-1 in control avr3x and pir-1 mutants, normalized to tubulin. (C-D) Bar graphs comparing abundance of small RNA species in reads per million (RPM) in arrested pir-1 mutants (7 days old) and L4 stage avr3x animals (as indicated). The error bar represents one standard error. P values were calculated for two replicas using unpaired student’s t-test (one-tailed for 22G-RNAs and 26G-RNAs, and two-tailed for miRNA). In (D), ‘n’ indicates the number of target genes in each category. (E) Histogram showing ratios of 22G-RNAs (pir-1/pir-1 + control avr3x) (x-axis) calculated for each individual gene in the two CSR-1 target categories and binned into 20 intervals plotted against frequency for each ratio (y-axis). See also Figure S4 and Table S3.

Figure 5.. Metagene analysis of 26G-RNA loci.

(A and B) Bar graphs plotting small RNA levels across mRNA intervals that template 26G-RNAs in the ALG-3/4 (A) and ERGO-1 pathways (B). Frequencies of mRNA-derived species (upper) and RdRP-derived species (lower) are plotted according to the position of their 5’ nt. Length is color-coded. Coordinates are defined relative to the C-nucleotide (–1) used to template 26G production. RNA was prepared from (fog-2) male-enriched populations (A) or WT embryos (B). See also Figure S5.

Figure 6.. pir-1 mutants are defective in 26G-RNA maturation.

(A) Bar graph comparing the levels of antisense 26G-RNAs (located at −1 in the metagene space) and sense-stranded 22mers (located at −23) cloned from of WT (avr3x), pir-1 null, and pir-1(C150S) respectively. Small RNAs were cloned using TAP or recombinant PIR-1 pretreatment to prevent cloning bias against ppp-RNA species (see Experimental Procedures). (B) Bar graph showing the ratio of 22mer to 26G-RNA in each strain using the data in panel (A). (C) Bar graph comparing the levels of p- and ppp-26G-RNA cloned from WT (avr3x) or pir-1(C150S) worms. Small RNAs were directly ligated to clone p-26G-RNAs (yellow), or they pretreated with recombinant PIR-1 to remove gamma and beta phosphates before ligation to clone p- and ppp-26G-RNAs (cyan). Reads were normalized to total 21U-RNAs. (D) Bar graph showing the ratio of ppp-26G-RNA to p-26G-RNA in WT and pir-1(C150S) data from (C). (E) Model of 26G-RNA biogenesis. P values were obtained using an unpaired Student’s t-test (one-tailed for A, B, and D; two-tailed for C) based on two replicas of each sample; error bars represent one standard error. See also Figure S6.