ceRNA Network Regulation of TGF-β, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta

Abstract

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-β, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3'UTR elements analyses of Tgf-β, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3'UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.

Keywords: FOXO; NGS; TGF-β; WNT; ascidian; miRNA; pseudogenes.

Figure 1

Functional classification with terms from Gene Ontology (GO) of the protein coding transcripts of C. robusta. The three GO classes are represented: (A): molecular function (MF), (B): biological process (BP), and (C): cellular component (CC). */** Chart tooltips are read as: category name (accession); * percent of gene hit against total # genes; ** percent of gene hit against total # function hits. Information is reported just for the three most representative subclasses for each GO class.

Figure 2

The left part of figure shows the whole pharynx transcripts of C. robusta. One percent of RNAs are noncoding. The right part of the figure shows the different classes of 1% of noncoding transcripts.

Figure 3

Multiple amino acid sequence alignment of Let7 miRNA family members from invertebrate, vertebrates and C. robusta.

Figure 4

Prediction of mRNA–miRNA and miRNA–pseudogene interactions through the miRNATip algorithm. In (A–C), (C.1,C.2) are represented respectively the steps of the used pipeline: (A) different transcripts produced by NGS were analyzed by miRNATIP predictor. (B) miRNATIP computes interactions of a couple of RNA molecules (miRNA–mRNA and miRNA–pseudogenes). The predictions of all interactions are then filtered by the user through an energy filter of <−12 of ΔG. (C.1) miRNA–mRNA target prediction results filtered for energy values. (C.2) miRNA–pseudogenes prediction results filtered for energy values.

Figure 5

Gene Ontology enrichment analysis of C. robusta transcripts which were predicted to interact with specific miRNAs and pseudogenes obtained by NGS sequences. All the three different Gene Ontology subcategories were investigated: (i) biological pathways (BP); (ii) molecular functions (MF); (iii) and cellular components (CC).

Figure 6

Analysis of 3′-UTR mRNA using the RegRNA web tool: MBE (musashi-binding element), GAIT (interferon-γ-activated inhibitor of translation), cytoplasmic polyadenylated element (CPE), MOS-PRE (polyadenylation response element), GU-rich destabilization element, RNA-binding protein (UNR), ARE (AU-rich element).

Figure 7

The four pathways are represented as networks formed by nodes connected by edges, and they are colored as follows: Wnt—green, Tgf-β—red and FoxO—yellow, Hh—purple. In RNA networks are also shown miRNAs and pseudogenes. Interacting ceRNAs are colored in green as they are interactors of Wnt pathway. The couple cin-mir-92c and ENSCING00000011392 is also ceRNA couple for HH2. Pink nodes represent proteins that are shared by two or more pathways.

Figure 8

ceRNA network reconstruction of Wnt, Tgf-β, FoxO and Hh pathways. Schematic representation of RNA-interacting molecules evidenced by miRNATIP algorithm is shown. Red arrow is inhibition, green arrow is activation, dashed line in indirect link. The same color in a ceRNA interacting couple (miRNA and pseudogene) indicate a direct interaction between the two RNA molecules.