Small RNAs regulate spermatogenesis in many species, ranging from Caenorhabditis elegans to mammals. In C. elegans, two Argonaute proteins, ALG-3 and ALG-4, and their associated alg-3/4 26G-small RNAs are essential for spermatogenesis at 25°C and for imprinting paternal memory of germline gene expression in offspring. The alg-3/4 26G-small RNAs are antisense to their target mRNAs and are known to be produced by the RNA-dependent RNA polymerase, RRF-3. However, it remains unclear how the RNA templates for RRF-3 are generated and which cellular processes are affected by alg-3/4 26G-small RNAs. Here, we demonstrate a key role for the conserved zc3h12a-ribonuclease-like NYN-domain-containing protein, NYN-3, in spermatogenesis at 25°C. Expression of NYN-3 was temporally coordinated with ALG-3, and our sequencing of both total and 2xFLAG::ALG-3-immunoprecipitated small RNAs revealed that NYN-3 is required for the biogenesis of alg-3/4 26G small RNAs. We further used ePAR-CLIP sequencing to identify NYN-3 binding sites on alg-3/4-targeted mature mRNAs; the NYN-3 binding sites were downstream of the binding sites for alg-3/4 26G-small RNAs. Additionally, 3'-RACE results placed the NYN-3 mRNA recognition upstream of RRF-3 binding and further revealed NYN-3 cleavage sites in alg-3/4-targeted mRNAs. Finally, a bioinformatics analysis was performed to parse the 26G small RNA-targeted genes into functional subclasses (e.g., signaling, chromatin defects). Collectively, these findings reveal NYN-3 as an initiator of small RNA generation that is central to the coordination of 26G small RNA-mediated gene regulation during spermatogenesis.

Total male and ALG-3 bound small RNA libraries:

The males from 25°C-grown gravid adult fog-2(q71); 2xFLAG::ALG-3 strains in various nyn-3 mutant backgrounds were first enriched using a 35-mm mesh. RNAs were either extract from enriched males or after performing immunoprecipitation (IP) using anti-FLAG. The extracted small RNAs were subjected to 15% (w/v) acrylamide/7M urea denaturing gel electrophoresis with TBE as the running buffer. RNAs ranging in size between 18 to 60 nt were gel purified and cloned, either with or without treatment of RppH (New England Biolabs [NEB], Ipswich, USA). The rest of the cloning procedures were performed as described previously, except the cloning primers were adjusted for the Illumina platform.

NYN-3 ePAR-CLIP-seq

One millimolar 4-thiouridine (Cayman Chemical, Ann Arbor, USA) was mixed in concentrated OP50 (an E. coli strain) and fed to L4 adults. After growth at 25°C overnight, HA-tagged WT and nyn-3 mutants with 2xflag::alg-3 were harvested as gravid adults, and the males were enriched by filtering through a 35-mm mesh (Spectrum Labs, New Brunwick, USA). The male-enriched population was irradiated with 365 nm UV light at 2 J/cm2 After the irradiation, the male-enriched worm pellet was mixed with one volume of iCLIP lysis buffer was used to prepare ePAR-CLIP samples. Eighty microliters of anti-HA antibody-conjugated beads (Sigma-Aldrich, St. Louis, USA) per 10 mg total lysate were applied to the samples and incubated at 4°C for 16 h. The wash procedures for ePAR-CLIP were according to the original protocol

 HA::NYN-3 bound 3’ RACE-seq

RNA extracted from HA IPs were ligated to DNA oligos at the 3′ end, with the 3′ consensus region of Illumina P5 sequence, which was pre-activated with Mth RNA Ligase (NEB). For the 3′ RACE analyzed on 22% 1xTBE PAGE, the same pre-activated 3′ ligation primer for small RNA cloning was used for smaller size PCR amplicons to improve resolution on the PAGE. The ligation procedure followed the non-ATP ligation step in small RNA cloning. Ligation products were reverse transcribed using a primer complementary to the ligated sequence. The nested PCR procedure was used to specifically amplify the cleavage products; cDNAs were amplified (20 cycles) using the first-strand cDNA primer and a forward-gene-specific primer that targeted a site slightly beyond 26G small RNA corresponding sequence. Since long adaptor sequences can be recognized and used in the Illumina machine, two consecutive PCRs were performed. First, a region between the 26G small RNA corresponding sequences and ligated sequence was amplified. Second, a PCR was performed to incorporate barcodes and the rest of the P5 and P7 sequences. The cycle number was first determined using previously described cycle-estimation procedures. The 3′ RACE products were then amplified according to the estimated Ct value, with the last 6 cycles using a specific barcode with full length Illumina sequence added to each sample. The final amplicons (140 to 300 bp) were excised, eluted and used as the input for Illumina sequencing.