Developmental regulation and functional prediction of microRNAs in an expanded Fasciola hepatica miRNome

Abstract

1ABSTRACTThe liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analysed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.CONTRIBUTION TO THE FIELDPrevious studies identified 89 micro (mi)RNAs (non-coding RNAs responsible for regulating gene expression) in the trematode parasite, Fasciola hepatica, and attached functional annotations to the miRNAs that are secreted by liver fluke in vitro. This study expands the known miRNA complement of F. hepatica by 40%, adding an additional 61 miRNAs, many of which appear Fasciola-specific. We used this expanded dataset to perform the first analysis of developmental miRNA expression across intra-mammalian parasites, showing clear developmentally regulated expression profiles across infectious metacercariae, juvenile and adult parasites, and extracellular vesicles secreted by adult parasites. We performed rigorous functional annotation of cellular miRNAs, correlating their expression with mRNA transcriptomes to identify potential roles for specific miRNAs in regulating expression of important proteases and nerve/muscle transcripts, all of which are important for parasite virulence. We also functionally analysed miRNAs secreted by adult parasites in terms of interactions with host transcripts; for the first time this analysis was supported by host transcriptome data, linking secreted miRNAs with mRNAs that are downregulated by fluke infection in sheep and cattle. This work contributes new data, insights and analyses to the field, providing a rich source of hypotheses for future experimental validation.

Transcriptomic analysis supports a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

“…This study identified 103 miRNAs in liver fluke, including 14 novel miRNAs (Sheet A in S9 Table ) and 89 miRNAs reported in previous studies on F . hepatica [ 38 , 64 – 67 ]. Recently, Herron et al .…”

“…Recently, Herron et al . [ 38 ] highlighted considerable redundancy across published miRNAs and moved to refine the current cohort of F . hepatica miRNAs by removing duplicate sequences of high similarity and retaining the longer sequence as individual miRNAs.…”

“…miRNAs were defined using the following criteria; present in at least 2 out of 3 replicates for either in vitro or in vivo transcriptomes, a minimum of 10 reads mapped to the mature sequence, at least 1 read mapped to the star sequence, a significant randfold value and a minimum miRDeep2 score of 5. miRNA naming was consistent with that presented in Herron et al . [ 38 ]. Differential expression of identified miRNAs was carried out using DESeq2 (version 1.28.1), with an adjusted p -value of 0.001 for significance.…”

Discovery of long non-coding RNAs in the liver fluke,Fasciola hepatica

“…Efforts to develop new treatments for liver fluke infections have been supported by the development of systems-level experimental resources over the past decade, including genome, transcriptome, proteome and functional genomics methodologies for F. hepatica (McVeigh et al, 2018). This increased volume and resolution of 'omics data has yielded insights into the non-coding (nc)RNAs of Fasciola genomes, where (mi)RNA complements are beginning to be clarified (Xu et al, 2012;Fontenla et al, 2015;Fromm et al, 2015;Guo and Guo, 2019;Ovchinnikov et al, 2020;Ricafrente et al, 2020;Herron, 2021;Hu et al, 2021), including a subset that appears to be secreted within extracellular vesicles (EVs). Transfer RNA fragments are the only other ncRNA to have been identified from F. hepatica transcriptomes (Hu et al, 2021), but eukaryotic transcriptomes contain a plethora of ncRNA biotypes.…”

Unpacking extracellular vesicles: RNA cargo loading and function

“…Beyond this diagnostic use, mRNAs loaded in EVs were also shown to be translatable in recipient cells in vitro, leading to the notion of EVs acting as an intercellular messaging system (Valadi et al., 2007). RNAs have been shown to be present in bacterial (Sjöström et al., 2015), fungal (Da Silva et al., 2015), insect (Lefebvre et al., 2016), parasitic (Herron et al., 2022), and plant EVs (Baldrich et al., 2019), as well as in mammalian EVs, suggesting that this messaging system is a conserved function. Indeed, further work has suggested that functional effects of EVs on the cellular proliferation and viability of recipient cell populations is mediated specifically by the loaded RNA, indicating a wide role for EV loaded RNA in both physiological and pathological scenarios (Eldh et al., 2010; Ratajczak et al., 2006).…”