Conservation of a microRNA cluster in parasitic nematodes and profiling of miRNAs in excretory-secretory products and microvesicles of Haemonchus contortus.

Henry Y Gu,William Weir,Alan D Winter,Thomas Tzelos,Eileen Devaney,Collette Britton,Tom N Mcneilly,Neil D Marks,Timothy G Geary

doi:10.1371/journal.pntd.0006056

Abstract

microRNAs are small non-coding RNAs that are important regulators of gene expression in a range of animals, including nematodes. We have analysed a cluster of four miRNAs from the pathogenic nematode species Haemonchus contortus that are closely linked in the genome. We find that the cluster is conserved only in clade V parasitic nematodes and in some ascarids, but not in other clade III species nor in clade V free-living nematodes. Members of the cluster are present in parasite excretory-secretory products and can be detected in the abomasum and draining lymph nodes of infected sheep, indicating their release in vitro and in vivo. As observed for other parasitic nematodes, H. contortus adult worms release extracellular vesicles (EV). Small RNA libraries were prepared from vesicle-enriched and vesicle-depleted supernatants from both adult worms and L4 stage larvae. Comparison of the miRNA species in the different fractions indicated that specific miRNAs are packaged within vesicles, while others are more abundant in vesicle-depleted supernatant. Hierarchical clustering analysis indicated that the gut is the likely source of vesicle-associated miRNAs in the L4 stage, but not in the adult worm. These findings add to the growing body of work demonstrating that miRNAs released from parasitic helminths may play an important role in host-parasite interactions.

Highlights

MicroRNAs were first discovered in the free-living nematode Caenorhabditis elegans and have been found in the cells of plants, animals and humans
Different species of parasitic worms release microRNAs, small non-coding RNA molecules, some of which are known to interact with host genes to alter the immune response
By small RNA library sequencing and bioinformatics using available genome sequence data, we identified a total of 192 miRNAs from H. contortus L3 and adult worms in a previous study [5]

Summary

Introduction

MicroRNAs (miRNAs) were first discovered in the free-living nematode Caenorhabditis elegans and have been found in the cells of plants, animals and humans These small RNAs are post-transcriptional regulators of gene expression and their dysregulation has been linked to a range of different pathologies including immune-related diseases [1] and cancer [2]. MiRNAs act by binding to complementary sequences often located in the 3’UTR of target genes in the context of the RNA Induced Silencing Complex (RISC) This results in the degradation of the mRNA and/or repression of translation [3]. H. contortus is an important parasite of small ruminants; it has a typical trichostrongyle life cycle in which sheep become infected by ingestion of infective third stage larvae (L3) These develop into L4 stages in the abomasum and into adult worms, which have a blood-sucking habit resulting in anaemia and, in acute cases, death

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