Diversity and Expression of MicroRNAs in the Filarial Parasite, Brugia malayi

Abstract

Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5–7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.

Functional analysis of microRNA activity in Brugia malayi

“…The expression of all six genes with putative miR-71 target sequences was higher in adult females than in microfilariae (Table 1). As the amount of miR-71 has previously been reported to be 5 – 7 times higher in microfilariae than in adult parasites (Poole et al, 2014), the finding that expression of all six genes was higher in adult parasites was consistent with the hypothesis that expression of these genes might be in part regulated by miR-71, suggesting that these mRNAs did indeed contain active miR-71 recognition sites in their 3’ UTRs.…”

“…Recent deep sequencing projects have revealed the presence of many miRNA homologues in nematode parasites (reviewed in (Britton et al, 2014)). These include Ascaris suum (Wang et al, 2011), Brugia pahangi (Winter et al, 2012), Dirofilaria immitis (Fu et al, 2013) and the human filarial parasite Brugia malayi (Poole et al, 2010, 2014). While some putative miRNAs in parasitic nematodes are highly conserved, a large proportion appear to be novel (Winter et al, 2012).…”

“…1B, C). miR-71 was chosen for this study as it is highly expressed in microfilariae (Poole et al, 2014) and adult female parasites (Winter et al, 2012). Deep sequencing analysis of the miRNA population demonstrated that miR-71 was one of the five most abundant miRNAs present in the isolated embryo preparations used in the transient transfection studies (T. R. Unnasch and L. McReynolds, unpublished data).…”

“…The amount of mRNA encoded by each of these genes was quantified by real time qPCR in microfilariae and adult female parasites. These stages were chosen for analysis as previous studies have indicated that miR-71 levels are 3 – 7 fold higher in microfilariae than in adult females (Poole et al, 2014). Total B. malayi RNA was extracted from 1 × 10 6 microfilariae and from five female adults provided by the Filariasis Research Reagent Resource Center (FR3, Division of Microbiology and infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA), using a Trizol-based method and RNA mini kit (Ambion, USA).…”

Neglected Tropical Diseases in the Post-Genomic Era

“…Deep sequencing of RNAs isolated from the blood of hosts infected with filarial nematodes allowed the identification of parasite-specific miRNAs for the human pathogen Onchocerca volvulus [120]. A similar strategy may be used in the case of Brugia malayi now that several miRNAs have been characterized [121]. It is worth noting that the lack of a reliable typing method is a major limiting factor in the fight for global elimination of lymphatic filariasis [7].…”