Introduction

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs encoding 22-nucleotide (nt) long RNAs that regulate target mRNAs in plants and animals. The first miRNA, lin-4, was identified in a genetic screening for mutations involved in developmental timing in the nematode Caenorhabditis elegans in 1993 (Lee et al. 1993). Surprisingly, lin-4 does not encode a protein, but a novel 22-nt small RNA, which regulates lin-14 translation through RNA–mRNA interaction, and plays an important role in developmental timing in C. elegans (Lee et al. 1993; Wightman et al. 1993). Seven years later, a second 22-nt small RNA of this type, let-7, a gene also involved in C. elegans developmental timing was discovered (Reinhart et al. 2000). let-7 regulates developmental timing in C. elegans by translational repression of lin-41 and hbl-1 through RNA–RNA interactions with their 3′ untranslated regions (Slack et al. 2000; Abrahante et al. 2003; Roush and Slack 2008).

The discovery of lin-4 and let-7 small regulatory RNAs was very exciting in the field of life sciences for several reasons. First, homologs of the let-7 gene were identified in other animals including humans (Lagos-Quintana et al. 2001). The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA. Second, this type of small RNAs regulated gene expression through specific base-paring with 3′ untranslated regions of target mRNAs (Lee and Ambros 2001; Grosshans and Slack 2002). Third, the mechanism of RNA interference (RNAi), which was mediated by small interfering RNAs (siRNAs) derived from cleavage of long endogenous or exogenous dsRNA, was discovered at that time, and it became clear that miRNA and RNAi pathways share common components (Banerjee and Slack 2002; Hutvagner and Zamore 2002; Tang and Zamore 2004).

Within the following 5 years from the discovery of let-7, more than 100 additional small regulatory RNAs similar to lin-4 and let-7 were identified in the worm C. elegans, the fruit fly Drosophila, and humans (Lau et al. 2001; Lai et al. 2003; Abbott et al. 2005; Bentwich et al. 2005). These small non-coding RNAs were named microRNAs. Subsequently, many more short regulatory RNAs were identified in almost all multicellular organisms, including plants (Rhoades et al. 2002), worms (Ambros 2003), flies (Ambros 2003), fish (Schier and Giraldez 2006), frogs (Tang and Maxwell 2008), and mammals (Smibert and Lai 2008) and in single cellular algae (Zhao et al. 2007) and viruses (Omoto and Fujii 2005; Simon-Mateo and Garcia 2006; Hussain et al. 2008). miRNAs are now considered as a key mechanism of post-transcriptional control within the networks of gene regulation.

Biogenesis of miRNAs

Although mature miRNAs are only about 22-nt long, their biogenesis is complicated. miRNAs are transcribed by RNA polymerase II in larger precursors as primary miRNAs (pri-miRNAs) containing characteristic stem–loop structures that are processed in the nucleus by a complex of the RNase III enzyme Drosha (Borchert et al. 2006). Drosha products are about 65-nt long hairpins called pre-miRNAs. pre-miRNAs are exported into the cytoplasm by Exportin-5 and Ran-GTP, where they are cleaved by the RNase III enzyme Dicer to release the mature miRNAs that are RNA duplexes of about 22-nt in length (Kim 2004). One miRNA strand of the duplex, called the miR strand, is selectively loaded onto an argonaute (AGO) protein, the RNA-induced silencing complex is formed and can now bind to, and repress target mRNAs containing sites of partially complementary to the miRNA (Ronemus et al. 2006; Ding et al. 2009). These miRNAs perform a variety of significant functions of cells such as in growth, metabolism, development, and cell differentiation (Wienholds and Plasterk 2005; Du and Zamore 2007).

miRNAs in protozoan parasites

miRNAs can regulate gene expression in eukaryotes at the post-transcriptional level. miRNAs exist in some protozoan parasites based on following considerations. First, this mechanism is very important for protozoan parasites, by which they could regulate gene expression in host cells to improve their abilities to infect and proliferate via inhibition of host immune responses, and change their gene expression to escape immunologic surveillance (Ong et al. 2006). Second, protozoan parasites belong to eukaryotes with big genome encoding many genes, and abundant antisense RNAs have been identified in certain species (Pascolo et al. 1993; Zamore 2002). Third, RNAi pathway sharing common components with miRNA is positive for some protozoan parasites (Lemos and Menezes 1978; Blackman 2003). Finally, the complex life cycles of many protozoan parasites require the ability to respond to environmental and developmental signals through regulating gene expression (Jolly et al. 2007).

In fact, the miRNA pathway requires AGO and Dicer proteins involved in its biogenesis. AGO- and Dicer-like proteins have been identified in Trypanosoma congolense, Leishmania braziliensis, Giardia lamblia, Entameoba histolytica, Trichomonas vaginalis, and Toxoplasma gondii by a comparative genomics approach (Best et al. 2005; Krautz-Peterson and Skelly 2008; Prucca et al. 2008). However, no AGO- and Dicer-like proteins have been found in the genomes of Leishmania major, Leishmania infantum, Trypanosoma cruzi, Plasmodium spp., Cryptosporidium spp., Theileria spp., Babesia bovis, and Eimeria tenella (Militello et al. 2008). Thus, some but not all protozoan parasites have the miRNA regulating pathway.

miRNAs in Trypanosoma brucei

T. brucei develops chronic infection in mammalian hosts due to antigenic variation. Most of the gene regulation takes place at the post-transcriptional level, which reflects the role of miRNAs in mRNA metabolism (Aitcheson et al. 2005). A total of 1,162 potential miRNAs have been found in T. brucei using bioinformatics approaches. The entire surface of the parasite is covered with a dense coat made up of approximately five million dimers of a single antigen, the variant surface glycoprotein (VSG). Individual trypanosomes have hundreds of VSG genes, but only one of which is expressed at a time. The predicted 16 miRNAs are considered to regulate expression of VSGs. Furthermore, a number of miRNA hairpins have been found in clusters of multiple identical copies. The target proteins, 20S proteosome, GM6, and GRESAG 4.2 corresponding to these clustered miRNAs, play essential role in trypanosomiasis (Berberof et al. 1996; To and Wang 1997). These miRNAs can act as genetic switches modulating host–parasite interaction and provide useful clue toward control of trypanosomiasis (Mallick et al. 2008).

miRNAs in E. histolytica

E. histolytica is an anaerobic protozoan parasite causing dysentery and liver abscess, and killing an estimated 2.5 million people around the world each year, mostly in tropical countries. Entamoeba requires controlled regulation of gene expression for switching between cyst and trophozoite forms not only for its survival inside and outside the host, but also for growth in the liver at high oxygen tension. The presence of miRNA machinery in E. histolytica inspired researchers to investigate the presence of putative miRNAs and their targets in this protozoan parasite (Abed and Ankri 2005; Solis and Guillen 2008). Seventeen putative candidate miRNAs in E. histolytica were recently identified using the bioinformatics approaches based on its genome (De et al. 2006). However, the putative miRNA candidates and their functions need further experimental validation.

miRNAs in G. lamblia

G. lamblia, one of the earliest branching eukaryotes, is a unicellular and binucleated protozoan responsible for giardiasis in humans. Gene expression is usually regulated at transcriptional and translational levels in higher eukaryotes. However, few consensus promoters have been identified and a simple AT-rich region was sufficient to initiate transcription in Giardia, and its mRNAs have exceedingly short 3′ and 5′-UTRs, thus greatly reducing the availability of regulatory sites for translational regulation (Adam 2001). Therefore, Giardia represents a unique model for studying the evolution of eukaryotic translational regulation.

The genome analysis showed no homologs of Drosha or Exportin-5 in Giardia, which is not necessary for miRNA pathway due to non-complete nuclear envelope of Giardia (Adam 2001). However, Dicer and AGO homologs were found in Giardia (Prucca et al. 2008). These data raised the possibility that miRNA-mediated translational repression could be one mechanism of gene regulation of this protozoan. Small RNAs of G. lamblia were cloned and sequenced, and four miRNAs derived from small nucleolar RNA (snoRNA) were identified, suggesting that the snoRNAs can be precursors of miRNAs in Giardia (Saraiya and Wang 2008). To date, miRNA precursors have been identified among non-coding cellular transcripts, 3′-UTRs of mRNAs, introns, transposable elements, and viral transcript (Cullen 2004; Klase et al. 2007; Piriyapongsa and Jordan 2007; Ruby et al. 2007). This unique phenomenon reflects the potential evolutionary significance of Giardia. The presence of a snoRNA-derived miRNA-mediated translational repression in Giardia has been demonstrated by increase and decrease of miR2 expression (Saraiya and Wang 2008). Additional 50 potential miRNAs, which are unique to G. lamblia, have been identified using computational programs (Zhang et al. 2009). This is the first report of cloning and experimental validation of miRNA in protozoan parasite, which provides a novel way for identification and functional analysis of miRNA in other parasites.

miRNAs in T. vaginalis

T. vaginalis, belonging to a highly diverged eukaryotic lineage, is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases. T. vaginalis contains 59,672 protein-coding genes, 1,136 RNA-coding genes, and 38,201 repeat genes (Aurrecoechea et al. 2009), the largest number of genes among all protozoan genomes, and there is a very stringent control of differentially expressed genes under variable environments, which makes it a very interesting model organism to study cellular processes.

The Trichomonas genome encodes at least two AGO proteins which contain AGO specific Piwi domains (Carlton et al. 2007). The expression of Tv-AGO1 (TVAG-453810) and Tv-AGO2 (TVAG-411040) genes were determined by quantitative real-time PCR, suggesting that functional miRNA machinery exists in T. vaginalis. Thereafter, a total of nine tva-miRNA (tva-miR-001∼009) ranging from 17 to 23 nucleotides long have been identified using direct cloning of miRNA tags and bioinformatics analysis (Lin et al. 2009). However, the exact length of the cloned miRNAs in T. vaginalis, target prediction and miRNA–target interaction are not yet known.

miRNAs in other protozoan parasites

Database mining of the predicted coding regions of T. gondii, T. congolense, and L. braziliensis revealed the existence of putative ORFs with convincing homology to the classical RNAi genes, namely potential homologues of AGO and Dicer, which are also the key enzymes in miRNA regulatory pathway (Militello et al. 2008). Furthermore, the expression of the T. gondii AGO-like protein (TgAGO) is supported by a number of tachyzoite cDNAs and exhibits unique features. For example, TgAgo is smaller than reported AGO proteins derived from higher eukaryotic organisms, but has a similar size to those from archaeal bacteria. TgAgo contains a conserved PiWi domain and non-conserved PAZ domain, and is mainly localized in the cytoplasm (Al Riyahi et al. 2006). However, the AGO-like proteins of T. congolense and L. braziliensis have not yet been identified.

However, AGO- and Dicer-like proteins have not been found in L. major, L. infantum, T. cruzi, Plasmodium spp., Cryptosporidium spp., Theileria spp., B. bovis, and E. tenella using bioinformatics analysis. Recently, the absence of miRNAs in Plasmodium falciparum has been experimentally validated by small RNA sequencing, which confirms with the absence of AGO/Dicer genes (Xue et al. 2008b). We can therefore conclude that the miRNA-mediated gene regulation may be absent in protozoan without AGO/Dicer proteins.

miRNAs in helminth parasites

Parasites of the class trematodes, especially Schistosoma mansoni and Schistosoma japonicum with a complex life cycle and a unique repertoire of genes expressed at different life cycle stages, are important eukaryotic pathogens of humans. miRNAs may be involved in growth, development, and cell differentiation of these parasites.

miRNAs in S. mansoni

Genomics and transcriptomics studies have revealed several ESTs in schistosomes with homology to Dicer and AGO, protein components of the miRNA silencing pathway. miRNA pathway in S. mansoni has recently been proven positive by bioinformatic approaches. A total of 13 putative proteins related to miRNA pathway in S. mansoni were found using amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases. These proteins participate in processing of miRNA precursor (SmDicer1), processing of primary miRNA transcripts (SmDrosha1/2), short RNA binding (SmAgo1/2/3/4), and nuclear export of miRNA precursors (Exportin-5-like protein; Gomes et al. 2009).

The expression of SmDicer1 and SmAgo2/3/4 transcripts is significantly different in different developmental stages of S. mansoni. The expression of SmDicer1 increases significantly from cercariae to schistosomula stage following the mechanical transformation. For SmAgo2/3/4 transcripts, a significant decreases from cercariae to MTS-8.5 followed by an increase in MTS-18.5 peaking at MTS-48 stage and then declining in the MTS-72. SmDicer1 and SmAgo2/3/4 transcripts reach their high expression levels in the egg stage (Krautz-Peterson and Skelly 2008; Gomes et al. 2009). In addition, various groups have used the RNAi technique to regulate gene expression in sporocysts, schistosomula, and adult worms (Boyle et al. 2003). All data have demonstrated that S. mansoni has miRNA-mediated gene regulation mechanism. However, the specific miRNAs in this parasite have not been reported.

miRNAs in S. japonicum

In S. japonicum, five novel miRNAs were identified and designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125, and sja-miR-new1. The expression patterns of these miRNAs are highly stage-specific, especially sja-miR-71 and sja-bantam, where expression reaches a peak in the cercaria stage and then drops quickly in the schistosomulum stage. These findings suggest that these miRNAs are involved in schistosome infection, growth, and development (Xue et al. 2008a). Further work is required to identify the target mRNAs of these novel miRNAs and elucidate the functions of newly identified miRNAs in S. japonicum.

miRNAs in arthropods

The mosquitoes Anopheles gambiae and Anopheles stephensi are the principal vectors of malaria in Africa and Asia. Understanding the functions of mosquito miRNAs will contribute to a better understanding of mosquito biology, such as longevity, reproduction, and mosquito–pathogen interactions, which are important to disease transmission.

About 91 candidate miRNAs, along with their pre-miRNAs, were identified in A. gambiae by searching the homologues of known Drosophila melanogaster miRNAs, in which 41 predicted miRNAs are known D. melanogaster miRNAs and the remaining 50 miRNAs are potential novel A. gambiae miRNAs (Chatterjee and Chaudhuri 2006). In A. stephensi, the four novel miRNAs (miR-x1-miR-x4) have been identified. The expression of miR-x2 is restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level is observed 72 h after a bloodmeal. Therefore, miR-x2 is likely to be involved in female reproduction and its function may be conserved among divergent mosquitoes (Mead and Tu 2008). A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day-old A. stephensi female mosquitoes. A. stephensi miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood-feeding status (Mead and Tu 2008). Thus, miR-14 is likely to be important across all mosquito life stages.

Potential functions of miRNAs in parasites

The discovery of miRNAs is one of the major scientific breakthroughs in recent years, and hundreds of miRNAs have been identified in various eukaryotic organisms. These tiny regulators of gene expression have unique tissue-, developmental-stage-, and disease-specific patterns. Although the functions of most miRNAs discovered have not been determined, primary functions of miRNAs suggest that miRNAs regulate some kinds of physiological and pathological processes in parasites.

miRNAs regulate a variety of developmental and physiological processes in parasites. For example, sja-let-7 might take part in the transformation from miracidium to sporocyst in the snail intermediate host; sja-bantam might take part in developmental processes throughout the lifespan of S. japonicum (Xue et al. 2008a); miR-14 is likely important across all mosquito life stages from embryos to aged adults (Mead and Tu 2008). Nevertheless, miRNAs may take part in pathogenic process of parasites. T. brucei miRNAs modulate expression of VSGs related to immune evasion, and T. brucei miRNA target proteins 20S proteosome, GM6, and GRESAG 4.2 are involved in trypanosomiasis (Mallick et al. 2008). In Giardia, miR2 may contribute to the pathogenicity by regulating expression of the 22 VSP genes (Saraiya and Wang 2008).

While parasite miRNAs can regulate their own gene expression, it is likely that parasite miRNAs also regulate expression of the host genes. Both parasites and hosts encode miRNAs, by which the parasites could make cellular environment highly susceptible to their propagation and survival, and hosts could make cellular environment unsusceptible to parasite survival. For example, let-7i regulates TLR4 expression in cholangiocytes and contributes to epithelial immune responses against Cryptosporidium parvum infection (Chen et al. 2007); knocking down Dicer1 and AGO1 mRNAs led to an increased sensitivity to Plasmodium infection (Winter et al. 2007).

Perspectives

To date, approximately 1,300 potential miRNAs have been identified in parasites and their number is still increasing. Due to the approaches used for their discovery, miRNAs with abundant and widespread expression have been found first. Further work aimed at identifying the target mRNAs of these novel miRNAs will be needed to interpret functions of newly identified miRNAs.

Because of the complex life cycles of parasites with several developmental stages in vertebrate and invertebrate hosts and a unique repertoire of genes expressed at different developmental stages, it is particularly important to elucidate the roles of miRNAs in the growth and development of parasites and their abilities to regulate infection of mammalian hosts. Clarifying of gene regulation based on miRNAs will contribute to dissection of the biological basis of antigenic variation and immune evasion for parasites.

Recent findings that antisense oligonucleotides can specifically block miRNA will trigger efforts to explore miRNAs as a potential new class of therapeutics (Zhang and Farwell 2008). We can anticipate the discovery of more and more cell-type- or developmental-stage-specific miRNAs with highly specialized functions related to the cellular processes in parasites in the coming years, which may provide novel therapeutics for parasitic diseases.