Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes

Abstract

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.

Keywords: dauer larvae; microRNA; nematodes; parasites; post-transcriptional regulation.

Fig. 1.—

Experimental setup and computational workflow. Life cycles of Caenorhabditis elegans, Pristionchus pacificus, and Strongyloides ratti. (A) Under favorable conditions for reproduction, larvae of C. elegans and P. pacificus develop through four larval stages. Under unfavorable environmental conditions, L2 larvae enter dauer diapause. (B) Infective larvae of S. ratti develop either directly or after facultative sexual free-living adult generation. Multiplatform small RNA deep sequencing was performed on mixed and dauer stage samples of C. elegans and P. pacificus. Illumina high-throughput profiling was carried out on mixed and infective stages of S. ratti. (C) Schematic overview of in house developed miRNA prediction pipeline. (D) miRNA gene complement in all three species, including novel gene candidates (note: several genes occur multiple times in the genome).

Fig. 2.—

miRNA homology and seed conservation. (A) miRNA preprocessing gives rise to two potential seed sequences (modified from Friedländer et al. 2008). (B) A total of 725 precursors were stratified into 399 gene families by sequence similarity of the full miRNA arm based on the most conserved arm. If both arms of a miRNA were annotated, the arm contained in the largest group (inferred from all 1,335 miRNA 5′- and 3′-arms) was considered. If group sizes were equal, the arm with the highest degree of conservation was considered (see definition of miRNA ages in supplementary methods, Supplementary Material online, for details). In case of equal conservation level, a miRNA arm was randomly chosen. (C) A total of 725 precursors were stratified into 374 seed groups by perfect seed sequence identity (positions 2–8) considering seeds based on the most conserved miRNA arm of a precursor. The miRNA arm was selected as explained earlier.

Fig. 3.—

Small RNA-seq expression profiles in Caenorhabditis elegans agree with qRT-PCR data. (A) Contingency table of expression fold changes of C. elegans dauer versus mixed stage obtained by Illumina small RNA deep sequencing compared with qRT-PCR data of dauer versus L2m from Karp et al. (2011) classified according to three categories (upregulated, downregulated, and unaffected). Expression fold changes of both data sets are significantly correlated (P = 1.1 × 10⁻⁵, χ² test). (B) Quantitative comparison of expression fold changes obtained by small RNA-seq and qRT-PCR experiments in C. elegans. Names of all miRNAs with a significant expression change of at least 2-fold in both experiments are displayed. Significance of differential miRNA levels in small RNA-seq data between mixed stage and dauer/iL3 was determined by a two-sided binomial test constrained on the total library sizes followed by correction for multiple testing (FDR < 0.05).

Fig. 4.—

mir-71 and mir-34 family miRNAs as cross-species candidate regulators in developmental arrest. MSAs for two candidate miRNA families mir-71 (A) and mir-34 (B) computed by LocARNA (Will et al. 2007). Multiple alignments were constrained to align at the seed sequence position of each individual miRNA. The seed (position 2-8) of miR-71 and miR-34 is marked with a red line. Arcs above the alignment represent secondary structure information. Arc colors encode the fraction of canonical paired bases. Alignment colors are annotated according to their agreement with the predicted secondary structure. Nucleotides that are base-paired according to the structure are colored in green and unpaired bases in red. If mutations have occurred but base-pairing potential is preserved, nucleotides are displayed in blue (dark blue for mutations in both bases and light blue for single-sided mutations). Unpaired nucleotides are colored in black and gaps in gray. Heatmaps represent miRNA gene expression by color where heatmap rows are ordered by the inferred phylogeny from the alignments. Arrows next to the miRNA in the heatmap plot denote significantly up- (↑) or downregulation (↓) in dauer/iL3.

Fig. 5.—

Expression conservation of miR-34 seed neighbors. The neighborhood of miRNA seeds was analyzed regarding expression changes in dauer and infective larvae. The neighborhood subnetwork of the mir-34 seed reveals conserved upregulation of all Pristionchus pacificus seed neighbors. Node color represents expression changes classified into upregulated (blue), downregulated (red), and unaffected (light gray). Barplot next to a node represents the number of times a miRNA seed was identified in a specific nematode. Note that mir-4933 of Caenorhabditis elegans was not measured in our data and is represented in the light gray pie.