microRNAs in Macrobrachium olfersii embryos: Identification, their biogenesis components and potential targets

Highlights

• Seven conserved and 10 novel miRNAs were identified in M. olfersii embryos.

• miRNA biogenesis components are evolutionarily conserved.

• In general, 516 miRNA targets were predicted in M. olfersii.

• Among the miRNAs identified, 10 novel miRNAs were Macrobrachium genus-specific.

Abstract

In embryonic development, microRNAs (miRNAs) regulate the complex gene expression associated with the complexity of embryogenesis. Today, few studies have been conducted on the identification of miRNAs and components of miRNA biogenesis on embryonic development in crustaceans, especially in prawns. In this context, the aim of this study was to identify in silico components of miRNA biogenesis, and miRNAs and potential target genes during embryonic development in the prawn Macrobrachium olfersii through small RNAs and transcriptome analyses. Using the miRDeep2 program, we identified 17 miRNA precursors in M. olfersii, which seven (miR-9, miR-10, miR-92, miR-125, miR-305, miR-1175, and miR-2788) were reported in the miRBase database, indicating high evolutionary conservation of these sequences among animals. The other 10 miRNAs of M. olfersii were novel miRNAs and only similar to Macrobrachium niponnense miRNAs, indicating genus-specific miRNAs. In addition, eight key components of miRNA biogenesis (DROSHA, PASHA/DGCR8, XPO5, RAN, DICER, TRBP2, AGO, and PIWI) were identified in M. olfersii embryos unigenes. In the annotation of miRNA targets, 516 genes were similar to known sequences in the GenBank database. Regarding the conserved miRNAs, we verified that they were differentially expressed during embryonic development in M. olfersii. In conclusion, this is the first study that identifies conserved and novel miRNAs in the prawn M. olfersii with some miRNA target genes involved in embryonic development. Our results will allow further studies on the function of these miRNAs and miRNA biogenesis components during embryonic development in M. olfersii and other prawns of commercial interest.

Introduction

MicroRNAs (miRNAs) are noncoding RNAs that play important roles in post-transcriptional gene regulation in animals and plants by degrading target messenger RNA (mRNA) or repressing targeted gene translation (Bartel, 2004; Moran et al., 2017). Typically, the mature miRNA binds to target sites at the 3′ UTR (untranslated region) of multiple target mRNAs in animals (Ambros, 2004), and a single mRNA can be targeted by one or more miRNAs (Felekkis et al., 2010; Shu et al., 2012). The biogenesis of miRNA starts with the transcription of primary microRNA (pri-miRNA), which is cleaved by the Drosha-DGCR8/Pasha complex to create precursor miRNA (pre-miRNA) (Bartel, 2004; Winter et al., 2009). The pre-miRNA is exported by Exportin-5-Ran-GTP to the cytoplasm and cleaved by the RNAse Dicer-TRBP complex. One strand of miRNA is incorporated into the RNA-induced silencing complex (RISC), using Argonaute proteins to silence target mRNAs (Winter et al., 2009).

The miRNAs have been identified in metazoa, mycetozoa, viridiplantae, and viruses (Sayed and Abdellatif, 2011) through computational or experimental approaches (Griffiths-Jones, 2006). A total of 35,828 miRNA genes have been annotated in the miRBase (release 21.0; June 2014; http://www.mirbase.org/). The identification of a large number of miRNAs is important because it allows researchers the opportunity to find miRNAs that are conserved between species or that are unique within species and to determine the function of conserved miRNAs in simpler organisms, as well as to discover more about the function of miRNAs in specific species (Lee et al., 2007; Li et al., 2010). Many miRNAs are evolutionarily conserved among a wide variety of multicellular organisms (Wienholds and Plasterk, 2005). Diverse biological functions for miRNAs have been described in normal development and physiological processes (Moran et al., 2017), including embryo formation, apoptotic cell death, cell proliferation, cell differentiation, metabolism, viral infection, and tumorigenesis (Bartel, 2009; Huang et al., 2011). For example, during the development of certain arthropod species (Drosophila melanogaster and Bombyx mori), many miRNAs exhibit temporal or tissue-specific patterns of gene expression related to possible regulatory roles, which has also been seen in studies involving other animals (Carrington and Ambros, 2003; Aboobaker et al., 2005; Liu et al., 2010a). The expression patterns of several fly miRNAs are analogous to those of their vertebrate counterparts, suggesting that these miRNAs may have ancient roles in animal patterning (Aboobaker et al., 2005). Thus, a large fraction of miRNAs in animals could play evolutionarily-conserved developmental or physiological roles (Carrington and Ambros, 2003).

The investigation into miRNAs and their biogenesis components of RNA interference (RNAi) machinery in non-classical species models, such as crustaceans, is important for elucidating their evolutionary pathways (Ikeda et al., 2015). Moreover, miRNAs are considered to be strongly related to the morphological evolution of animals (Ikeda et al., 2015). In crustaceans, the study of miRNAs is also important to determine their roles in immune defense mechanisms for disease control (He et al., 2015), to know the molecular basis of development and responses to environmental stimuli (Chen et al., 2014), and also to elucidate the evolution of miRNAs (Ikeda et al., 2015). The identification of miRNAs has been limited to a few species of crustaceans, such as Marsupenaeus japonicus, Litopenaeus vannamei, Procambarus clarkii, and Cherax quadricarinatus, mainly related to the miRNAs response to viral and bacterial infections (Huang et al., 2012a; Ou et al., 2013; Xi et al., 2015; Zeng et al., 2015; Zhao et al., 2016). Also, miRNAs have been described in the ovary of Eriocheir sinensis (Song et al., 2014), in the gills and hepatopancreas of Macrobrachium rosenbergii (Tan et al., 2013), and in different tissues of Macrobrachium nipponense (Jin et al., 2015; Sun et al., 2016). In relation to development, miRNAs have been identified in Parhyale hawaiensis (Blythe et al., 2012), Daphnia pulex (Chen et al., 2014) and Caligus rogercresseyi (Gallardo-Escárate et al., 2017). However, considering the great diversity of crustaceans, little information is available about the identification and function of miRNAs during developmental stages in these animals.

Macrobrachium olfersii (Crustacea; Decapoda; Palaemonidae), a freshwater prawn with geographical distribution in the Americas (Holthuis, 1952), presents interesting features (described in Jaramillo et al., 2016) that qualify this species as an emerging model for developmental and toxicological studies in crustaceans (Nazari et al., 2010, Nazari et al., 2013; Zeni et al., 2015; Jaramillo et al., 2017; Quadros et al., 2016; Schramm et al., 2017). In addition, the morphological and chronological characteristics of the embryonic development of this species are well known (Müller et al., 2003, 2004), and studies on the molecular basis of its development have been initiated in order to increase its comparability with other decapods. The embryonic development of M. olfersii occurs in 14 days at 24 °C (Simões-Costa et al., 2005), growing from a meroblastic superficial cleavage in a short germ-band development pattern, which does not allow the tracing of early cell lineages (Wolff and Scholtz, 2002). This feature of M. olfersii embryonic development differs from other crustaceans, for which information on miRNAs is available, such as for P. hawaiensis, which have holoblastic cleavage that allows tracing of early cell lineages (Browne et al., 2005; Alwes et al., 2011; Nestorov et al., 2013) and species of the genus Daphnia, which have meroblastic cleavage and long germ-band development types (Scholtz and Wolff, 2013).

Recently, transcriptome analysis and the identification of reference genes for RT-qPCR in embryos of M. olfersii have been performed (Jaramillo et al., 2016, 2017). However, the molecular aspects of the developmental mechanisms of this species are still unknown, and the miRNAs have not yet been described. This essential knowledge will allow us to combine classical embryology with new approaches in developmental biology, and thus, increase our understanding of the diversity and complexity of crustacean embryonic development. In this context, the aim of this study was to identify miRNAs, components of miRNA biogenesis, and miRNA target genes during embryonic development in M. olfersii. Our results describe for the first time miRNAs in M. olfersii that could contribute to future research on miRNA function during the development of M. olfersii and other prawns.