Small non-coding RNAs have predicted roles in reproductive biology and transposable element regulation in the parasitic worm Strongyloides venezuelensis

Abstract

The gastrointestinal parasitic nematode Strongyloides spp. has a unique life cycle that alternates between a parasitic generation that reproduces through mitotic parthenogenesis and a dioecious free-living sexually reproducing generation. Adult females from these two generations are genetically identical, making them an informative model to identify molecular differences between parasitic and free-living lifestyles and understand different reproductive strategies. We investigated the expression of small RNAs (sRNAs) that are either enriched for a 5' monophosphate modification (5'pN) or are 5' modification-independent, across five life cycle stages of the rodent parasite Strongyloides venezuelensis. We identified miRNAs and small-interfering RNAs expressed by S. venezuelensis that are predicted to target and regulate the expression of protein-coding genes and transposable elements (TEs). Three previously unreported classes of sRNA were identified: (1) 25Gs with a putative role in reproduction in adult females, (2) tRNA-derived 24-28 nt sRNAs (tsRNAs) which are predicted to target TEs in free-living females, and (3) 5'pN-enriched 26-29Cs with 5' CGAATCC and 3' TTT motifs expressed in parasitic females. We also confirmed that S. venezuelensis expresses the 27G class of sRNAs involved in TE regulation, which was previously identified in the rodent parasite Strongyloides ratti. Taken together, these results provide new insights into the role of sRNAs in reproductive biology and parasitism.

Keywords: Strongyloides venezuelensis; Helminth; Nematode; Parasite; Small RNA; microRNA.

Fig. 1

The Strongyloides life cycle stages used for small RNA analysis. Small RNA was sequenced from five life cycle stages of S. venezuelensis (yellow star): free-living female adults, free-living infective larvae (iL3) searching for a host, parasitic ‘activated’ infective larvae (activated iL3) in the host environment, adult parasitic females from the host’s intestine, and their eggs. Unlike other Strongyloides species, free-living males were not found in S. venezuelensis.

Fig. 2

Expression profiles of sRNAs from RppH-treated and untreated libraries. sRNA expression based on RPM (reads per million) in the RppH-untreated (5’pN-enriched) and treated libraries for two to three replicates of (A) free-living iL3s, (B) activated iL3s, (C) eggs, (D) parasitic females and (E) free-living females, collected from parasitic females. Classification of the sRNA based on the type of sequence the sRNA originates from in the genome (solid bars) and the first 5’ base in the sequence (hashed bars) are highlighted by colour. sRNAs are divided into either miRNA, rRNA-derived sRNA (rRNA), tRNA-derived sRNA (tRNA) or as putative siRNA originating from either protein-coding genes (CDS or intronic regions), 5’- and 3’-UTRs, intergenic regions or transposable elements (TE).

Fig. 3

sRNA upregulated in parasitic and free-living adults. sRNAs differentially expressed in free-living and parasitic females (FDR ≤ 0.01, edgeR Fishers Exact test when compared to ‘activated’ iL3s, free-living iL3s and eggs) in (A) RppH-treated library and (B) 5’pN-enriched library. Starting sequence base shown in key and proportion of origin (derived from tRNA, miRNA, rRNA, 500ds (3’UTR), 200us (5’UTR), TEs, intergenic regions or genes) of the expressed sRNAs shown below graphs.

Fig. 4

Gene Ontology (GO) enrichment analysis of free-living 25G gene targets. (A) Enriched GO terms associated with the predicted gene targets (the coding sequence (CDS) and 3’UTRs of protein coding genes) of 25G sRNAs upregulated in free-living females, parasitic females and non-differentially expressed sRNAs.. Enriched GO terms were visualised using REVIGO. Corresponding upregulated sRNAs first base, origin and nucleotide profile shown below. (B) the proteins associated with the free-living upregulated 25Gs. (C) Difference in expression level (RPKM: FLF minus PF) of genes putatively targeted by free-living upregulated, parasitic upregulated and non-DE 25Gs with a logFC of ≤ 1.5. RPKM = Reads per kilobase per million.

Fig. 5

sRNA expression of free-living and parasitic 18–30 nt long sRNAs with a 5’ monophosphate. (A) Number of 18–30 nt long sRNAs upregulated in parasitic females and free-living females compared with other stages in 5’pN-enriched libraries. Upregulated sRNAs were classified by the gene locations which those originated from (intergenic regions, TE, genes, 5’UTR, 3’UTR, rRNAs or tRNA). (B) Number of tsRNA upregulated in free-living and parasitic females, classified into types of tRNA they originate from.

Fig. 6

Nucleotide composition of parasitic and free-living 26–29Cs. The parasitic female upregulated 26–29Cs exhibited conserved CGAAUCC and UUU motifs at their 5’ and 3’ ends, respectively, which are absent in the free-living upregulated 26-29Cs. Visualised in WebLogo. A complete figure illustrating 26-29Cs can be found in SI Fig. 4.N = number of unique sequences.

Fig. 7

27Gs upregulated in parasitic and free-living adults. (A) Number of unique 27Gs upregulated in parasitic female against the four other life cycle stages. n = number of unique sequences upregulated in parasitic female against each life cycle stage. (B) Number of unique 27Gs upregulated in free-living female against the 4 other life cycle stages. n = number of unique sequences upregulated against each life cycle stage.

Fig. 8

sRNA upregulated in eggs, activated iL3s and free-living iL3s. sRNAs differentially expressed in activated iL3s, iL3s and eggs (FDR ≤ 0.01, edgeR Fishers Exact test when compared to other life cycle stages) in (A) RppH-treated library and (B) monophosphate library. Starting sequence base shown in key and proportion of origin (derived from tRNA, miRNA, rRNA, 500ds (3’UTR), 200us (5’UTR), TEs, intergenic regions or genes) of the expressed sRNAs shown below graphs.