DOI: 10.5061/dryad.b8gtht7ft
title: Biogenesis of C. elegans spermatogenesis small RNAs is initiated by a zc3h12a like ribonuclease

description: 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.
description: <strong>Total male and
 ALG-3 bound small RNA libraries:</strong>
 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.
 <strong>NYN-3
 ePAR-CLIP-seq</strong> One millimolar 4-thiouridine (Cayman Chemical,
 Ann Arbor, USA) was mixed in concentrated OP50 (an <em>E.
 coli </em>strain) and fed to L4 adults. After growth at
 25°C overnight, HA-tagged WT
 and <em>nyn-3</em> mutants with <em>2xflag::alg-3</em> 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 <strong> HA::NYN-3 bound 3’ RACE-seq</strong> 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 <em>Mth</em> 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.