Discovery of putative long non-coding RNAs expressed in the eyes of Astyanax mexicanus (Actinopterygii: Characidae)

Abstract

Astyanax mexicanus is a well-known model species, that has two morphotypes, cavefish, from subterranean rivers and surface fish, from surface rivers. They are morphologically distinct due to many troglomorphic traits in the cavefish, such as the absence of eyes. Most studies on A. mexicanus are focused on eye development and protein-coding genes involved in the process. However, lncRNAs did not get the same attention and very little is known about them. This study aimed to fill this knowledge gap, identifying, describing, classifying, and annotating lncRNAs expressed in the embryo's eye tissue of cavefish and surface fish. To do so, we constructed a concise workflow to assemble and evaluate transcriptomes, annotate protein-coding genes, ncRNAs families, predict the coding potential, identify putative lncRNAs, map them and predict interactions. This approach resulted in the identification of 33,069 and 19,493 putative lncRNAs respectively mapped in cavefish and surface fish. Thousands of these lncRNAs were annotated and identified as conserved in human and several species of fish. Hundreds of them were validated in silico, through ESTs. We identified lncRNAs associated with genes related to eye development. This is the case of a few lncRNAs associated with sox2, which we suggest being isomorphs of the SOX2-OT, a lncRNA that can regulate the expression of sox2. This work is one of the first studies to focus on the description of lncRNAs in A. mexicanus, highlighting several lncRNA targets and opening an important precedent for future studies focusing on lncRNAs expressed in A. mexicanus.

Figure 1

Simplified workflow of the conducted analysis, divided into three major steps. (1) Pre-processing of RNA-seq libraries, transcriptome assembly, quality assessment and general metrics; (2) Functional annotation of the transcriptomes, coding potential calculation, identification of ncRNAs families and filtering process. (3) Mapping of putative lncRNAs, identification of shared, novel, known and conserved lncRNAs, as well their classification, interactions and in silico validation of them through ESTs.

Figure 2

Number of sncRNAs, non-coding transcripts and the distribution of lncRNAs per chromosome. (a) Distribution of miRNAs and snoRNAs annotated in INFERNAL’s cmscan module; (b) Classification of transcripts according to their coding potential, considering the agreement of at least 4/5 coding potential tools; (c) Comparison of the chromosomal distribution of mapped lncRNAs between cavefish and surface fish.

Figure 3

Shared lncRNAs, number of annotations against the databases and the number of novel and conserved lncRNAs. (a) Venn diagram showing lncRNAs that are shared between the two morphotypes; (b) Number of lncRNAs annotated to AmexLNC, ZFLNC and LncBook databases; Venn diagram showing lncRNAs of (c) cavefish and (d) surface fish and how the annotations were distributed across the databases. The number of lncRNAs annotated against more than one database is represented in the intersections; (e) Dendrogram illustrating the lncRNAs of CF and SF that were conserved among other fishes species with assemblies available in Ensembl; (f) Number of lncRNAs that were not annotated to any lncRNA database (Novel LncRNAs) and lncRNAs that were annotated to at least one database (Known LncRNAs), therefore, conserved lncRNAs.

Figure 4

LncRNAs classification and interaction networks of lncRNAs and partners. (a) Distribution of intergenic lncRNAs, organized into three types: same-strand, convergent and divergent. Same-strand and convergent lincRNAs were the most abundant in both cavefish and surface fish, however, in cavefish, the number was considerably higher; (b) Distribution of genic lncRNAs, where cavefish had a higher number of intronic and surface fish of exonic; Interaction networks between lncRNAs of (c) cavefish and (d) surface fish and candidate genes (partners). The ‘DN’ prefix from the IDs of the lncRNAs was removed to enable better visualization. Moreover, to guarantee the legibility of these high-information figures, high-resolution versions of the interaction networks are available in Supplementary File 2 and 3, respectively; (e) Cavefish and (f) Surface fish lncRNAs, represented by outer circles, interacting with sox2 gene in the center. The tree circles with a background color other than white, represent homologous lncRNAs between morphotypes.

Figure 5

Comparison of secondary structures of RBH pairs of SOX2-OT transcripts. Motif colors represent conserved regions between the pair’s structures. (a) DN21854_c0_g1_i13 (CF) and DN1425_c0_g1_i7 (SF); (b) DN21854_c0_g1_i2 (CF) and DN1425_c0_g1_i27 (SF) and (c) DN21854_c0_g1_i10 (CF) and DN1425_c0_g1_i19 (SF).