Small RNA pathways in the nematode Ascaris in the absence of piRNAs

Abstract

Small RNA pathways play key and diverse regulatory roles in C. elegans, but our understanding of their conservation and contributions in other nematodes is limited. We analyzed small RNA pathways in the divergent parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that associate with secondary 5'-triphosphate 22-24G-RNAs. These small RNAs target repetitive sequences or mature mRNAs and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Even in the absence of a piRNA pathway, Ascaris CSR-1 may still function to "license" as well as fine-tune or repress gene expression. Ascaris ALG-4 and its associated 26G-RNAs target and likely repress specific mRNAs during testis meiosis. Ascaris WAGO small RNAs demonstrate target plasticity changing their targets between repeats and mRNAs during development. We provide a unique and comprehensive view of mRNA and small RNA expression throughout spermatogenesis. Overall, our study illustrates the conservation, divergence, dynamics, and flexibility of small RNA pathways in nematodes.

Fig. 1. Ascaris has 10 Argonautes.

A Argonaute protein phylogenetic tree showing the relationship of Ascaris and C. elegans Argonautes. Ascaris Argonautes are in purple boxes. Labels indicate the type of Argonaute or its associated small RNA. GenBank accession numbers for these Argonaute protein sequences are available in Supplementary Table 1. Argonautes highlighted in green represent a canonical fly PIWI and Arabidopsis AGO Argonaute. The Argonautes were aligned using Muscle and the tree generated using Neighbor Joining and Maximum Likelihood (1,000 bootstraps) with the bootstrap values illustrated. Ascaris proteins for which antibodies were generated are marked with Ab. B RNA expression of Ascaris Argonautes and RNA-dependent RNA polymerases (RdRPs). The levels of expression (numbers are in rpkm) are shown in a heatmap with higher expression in red. Note that we view an rpkm value that is less than 5 as a background level of expression. The developmental stages are as follows: M1 to M8 are regions of the male germline, with M1 = early mitotic region, M2 = late mitotic region, M3 = transition zone, M4 = transition zone to early pachytene, M5 = pachytene, M6 = late pachytene, M7 = meiosis diplotene to diakinesis, and M8 = spermatids (see Fig. 3, text, and Materials and Methods for a detailed description of the male germline stages); F1–F5 are regions of the female germline, with F1 = mitotic region, F2 = early pachytene, F3 = late pachytene, F4 = diplotene, F5 = oocyte; zygote maturation stages prior to pronuclear fusion isolated from the uterus (E1–4; E4 is the stage passed from Ascaris and the host to the environment); embryo development (at 30 °C) stages, with E5 = 24 h (1-cell), E6 = 46 h (2-cell), E7 = 64 h (4-cell), E8 = 96 h (16-cell), E9 = 116 h (32–64-cell), E10 = 7day (256-cell), larvae L1 (10-day) and L2 (21-day); and adult somatic tissues, with Mu Muscle, In intestine, and Ca carcass, which includes the cuticle, hypodermis, muscle, nervous system, and pharynx. The RNA-seq expression data are provided as a Source Data file. C Ascaris Argonaute immunohistochemistry in early embryos. DAPI (blue) in left panel and immunohistochemistry (green) in right panel. Data are derived from three or more biological replicates. Scale Bars = 10 µm.

Fig. 2. Ascaris Argonautes bind specific sets of small RNAs.

A Size distribution, frequency, and targets of small RNAs associated with specific Argonautes. Small RNAs (18–30 nt) from input and Argonaute immunoprecipitation (IP) were plotted for the whole testis (male germline), whole ovary (female germline), and 4-cell embryo (60 h). In these and all subsequent small RNA size distribution figures, small RNAs starting with A, C, G, and U of different sizes (x-axis) were plotted against their read frequencies (y-axis; raw reads in millions). Data are derived from two or more biological replicates combined. The small RNAs were categorized by their type (miRNA) or complementarity to different targets as shown in percentage bars below each size distribution plot. The target categories are sequences matching: (1) rRNAs or (2) tRNAs; (3) miRNAs; (4) repetitive sequences and mobile elements that are targets of AsWAGO-1, AsWAGO-2, and AsNRDE-3 (WAGO-repeats); (5) siRNAs antisense to mRNAs; siRNAs matching (6) introns or (7) intergenic regions; (8) small RNAs that have no full-length match to the genome (no match); (9) and sense small RNAs that correspond to mRNAs. B, C Principal component analysis (PCA) of small RNAs associated with Argonautes in different stages showing the overall relationship among these small RNA libraries. Panel B is PCA analysis of small RNAs that target mRNAs and C is small RNAs that target the WAGO-repeats. D, E Venn diagram showing the Argonaute small RNA overlapping targets in the testis for mRNAs (D) and WAGO-repeats (E). Source data are provided as a Source Data file.

Fig. 3. Ascaris male gonad regions and nuclear morphology.

A Schematic representation of regions of Ascaris male germline illustrated that correspond to C. elegans male germline and nuclear morphology. Corresponding regions between C. elegans and Ascaris are kept to scale; however, the length of the Ascaris male gonad is ~1 m (see Supplementary Fig. 3). Regions labeled are: (1) mitotic (pink), (2) transition zone (orange), (3) meiosis 1—pachytene, (4) meiosis 1 and 2 (green), and (5) spermatids (blue). Regions of the Ascaris male germline collected for Argonaute IP and RNA-seq are labeled M1–M8, separated by blue vertical lines. B Nuclear morphology of DAPI-stained regions of the Ascaris male gonad. Scale bars = 10 µm. a–d Overview of the mitotic region illustrating the progressive increase in gonad thickness. Mitotic stages are observed (metaphase, anaphase; arrows) and were confirmed by staining with CENP-A and the mitosis marker H3S10P, as well as apoptotic nuclei (*). Larger and less condensed nuclei are also detected, scattered through the mid-plane (dotted circles). e The transition zone exhibits more condensed and punctate nuclear morphology. f–h “Spaghetti bowl”-like nuclei characteristic of early (f), middle (g), and late pachytene (h). i–m Stages of meiotic progression: diplotene (i); diakinesis, with distinguishable individual bivalents (j); metaphase I (k); anaphase I (l), note lagging sex chromosomes (inset, arrowhead); and second meiotic division resulting in 4 haploid nuclei (m, contour). (n) spermatids. Data are derived from two or more biological replicates.

Fig. 4. Small RNAs associated with Ascaris Argonautes during spermatogenesis.

A Illustration of Ascaris stages of spermatogenesis from Fig. 3. B RNA expression profiles of Ascaris Argonautes and RdRPs throughout spermatogenesis. The Argonautes are plotted in two groups based on their expression pattern or their targets. The RNA-seq expression data are provided as a Source Data file. C Small RNAs from mature spermatids (M8). The random size distribution pattern illustrates the low levels of bona fide small RNAs. D Small RNAs during spermatogenesis (M1–M7). In C and D, the size distribution, frequency, and classification of small RNAs are as described as in Fig. 2A.

Fig. 5. AsWAGO-1, AsWAGO-2, and AsNRDE-3 associated small RNAs target repeats during spermatogenesis.

A Genome browser view of a region of chromosome 1 illustrating Argonaute associated small RNAs, RNA expression (RNA-seq), and H3K9me3 levels. Only spermatogenesis stages that exhibit changes in expression or changes in Argonaute small RNA or mRNA expression are illustrated. Note NRDE-3 associated small RNAs change their targets from repeats to mRNAs during pachytene and late meiosis (M5–M7). B Ascaris WAGO-repeat targets largely overlap with genomic H3K9me3 levels and/or the presence of transposable elements. C siRNA levels to the top targets (top 1776 WAGO-repeat loci; see Supplementary Table 3) for each WAGO during spermatogenesis. Heatmaps illustrate the standard Z-score (converted from rpkm) showing changes in expression of siRNAs associated with AsWAGO-1, AsWAGO-2, and AsNRDE-3. The RNA expression (rpkm) of the targets is also illustrated (right). Targeted loci were sorted based on the same order in the four heatmaps. Source data are provided as a Source Data file.

Fig. 6. AsCSR-1 and AsNRDE-3 license or fine-tune mRNA expression during early spermatogenesis whereas AsCSR-1, AsNRDE-3, and AsALG-4 facilitate the clearance of mRNAs during late spermatogenesis.

A Genome browser view of a region of chromosome 1 illustrating AsCSR-1, AsNRDE-3, and AsALG-4 associated small RNAs and their mRNA target expression during spermatogenesis. B AsCSR-1, AsNRDE-3, and AsALG-4 mRNA targets during spermatogenesis. AsCSR-1 small RNAs targets are pervasive in different stages and correlate with RNA expression, AsNRDE-3 small RNA targets switch from repeats to mRNAs in pachytene and late meiosis (M5–M7), whereas AsALG-4 is expressed and its associated 26G-RNAs target mRNAs that are primarily restricted to meiosis (M6–M7) in the male gonad. Shown are the number of mRNAs targeted by different Argonautes. The color indicates where these mRNA targets are most highly expressed in developmental stages using the same color scheme as Fig. 1B. Note AsCSR-1 and AsNRDE-3 target a broad group of mRNAs, while AsALG-4 targets mostly testis-specific genes. C AsCSR-1, AsNRDE-3, and AsALG-4 targeted mRNAs largely overlap during late meiosis. Venn diagrams showing the relationship between these targets in the M6 stage where all three Argonautes have a large number of targets. D siRNA and targeted mRNA levels in M6 (Fig. 6C) during spermatogenesis. Very low levels of small RNAs are in mature spermatids (Fig. 4C); thus, a 0 value is used in M8 for the AGO IPs. Heatmaps illustrate the standard Z-score (converted from rpkm) showing changes in siRNA and mRNA expression. Targeted genes were sorted based on the same order in the heatmap pairs. E AsNRDE-3 siRNAs are enriched for sequence at the 5’ ends of mRNAs. siRNA distribution on mRNAs changes in AsNRDE-3, AsCSR-1, and AsALG-4 during spermatogenesis (see also Supplementary Fig. 6). Source data are provided as a Source Data file.

Fig. 7. Plasticity of Ascaris small RNA pathways.

A AsNRDE-3 bound small RNAs change their targets from genomic repetitive sequence to mRNAs during spermatogenesis (M5–M7). B AsNRDE-3 mRNA and repeat target changes by during spermatogenesis. C WAGO-3 bound small RNAs change their targets from genomic repetitive sequence in male mitotic germline to mRNAs in 4-cell embryos. Source data are provided as a Source Data file. D Model for Ascaris small RNA pathways (see text). The two concentric circles represent Ascaris mRNAs (left) and repeats (right) targeted by small RNAs. The larger size of the mRNA circle indicates the larger number, complexity, and abundance of the mRNAs compared to the repeats targeted by small RNAs. Overall, however, in terms of abundance of small RNAs, 80–90% of all small RNAs target repeats. The 10 Ascaris Argonautes and their known associated major small RNAs (22 G, 23 G, 24 G, 26 G, and miRNAs) are shown. Lines with an arrow indicate licensing or fine-tuning (→) of expression, a blocking line indicates repression (^__|), and an arrow with a blocking line indicates both licensing/tuning and repression (→|). Bolder lines indicate predicted stronger affects. Note several Ascaris Argonautes (CSR-1, WAGO-3, and NRDE-3) could be involved in both licensing and repression of their mRNA targets. In addition, NRDE-3 and WAGO-3 can target both mRNAs and repeats and their targets change through development, illustrating the plasticity of small RNA pathway in Ascaris.