Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

stepRNA: Identification of Dicer cleavage signatures and passenger strand lengths in small RNA sequences.

The enzyme Dicer is a component of many small RNA (sRNA) pathways involved in RNA processing for post-transcriptional regulation, anti-viral response and control of transposable elements. Cleavage of double-stranded RNA by Dicer produces a signature overhanging sequence at the 3' end of the sRNA sequence relative to a complementary passenger strand in a RNA duplex. There is a need for reliable tools to computationally search for Dicer cleavage signatures to help characterise families of sRNAs. This is increasingly important due to the rising popularity of sRNA sequencing, especially in non-model organisms. Here, we present stepRNA, a fast, local tool that identifies (i) overhang signatures strongly indicative of Dicer cleavage in RNA sequences, and (ii) the length of the passenger strand in sRNAs duplexes. We demonstrate the use of stepRNA with simulated and biological datasets to detect Dicer cleavage signatures in experimentally validated examples. Compared to currently available tools, stepRNA is more accurate, requires only sRNA sequence data rather than a reference genome, and provides information about other important features such as passenger strand length. stepRNA is freely available at https://github.com/Vicky-Hunt-Lab/stepRNA and is easily installable.

piRNA-like small RNAs target transposable elements in a Clade IV parasitic nematode.

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C. elegans group (Clade V), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21-22 nt long sRNAs with a 5' uridine (21-22Us) and a 5' monophosphate, and (ii) 27 nt long sRNAs with a 5' guanine/adenine (27GAs) and a 5' modification. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome. Overall, we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V.