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Biology (Basel)
2019 Aug 24;83:. doi: 10.3390/biology8030061.
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Microsyntenic Clusters Reveal Conservation of lncRNAs in Chordates Despite Absence of Sequence Conservation.
Herrera-Úbeda C
,
Marín-Barba M
,
Navas-Pérez E
,
Gravemeyer J
,
Albuixech-Crespo B
,
Wheeler GN
,
Garcia-Fernàndez J
.
Abstract
Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to their fast-evolutionary rate. Here we develop LincOFinder, a pipeline that finds conserved intergenic lncRNAs (lincRNAs) between distant related species by means of microsynteny analyses. Using this tool, we have identified 16 bona fide homologous lincRNAs between the amphioxus and human genomes. We characterized and compared in amphioxus and Xenopus the expression domain of one of them, Hotairm1, located in the anterior part of the Hox cluster. In addition, we analyzed the function of this lincRNA in Xenopus, showing that its disruption produces a severe headless phenotype, most probably by interfering with the regulation of the Hox cluster. Our results strongly suggest that this lincRNA has probably been regulating the Hox cluster since the early origin of chordates. Our work pioneers the use of syntenic searches to identify non-coding genes over long evolutionary distances and helps to further understand lncRNA evolution.
Figure 1. Diagram of LincOFinder mechanism. (A) Representation of the Ref species region where a lincRNA is present. (B) Formatted table of orthologs and virtual coordinates from the three upstream and downstream coding genes fed to the algorithm. (C) Selection of the best cluster according to the minimum distance between genes. (D) Representation of a conserved mycrosyntenic cluster in the Int species, where the presence of a lincRNA is manually confirmed (above) or discarded (below).
Figure 2. Schematic representation of the genomic locus of Hotairm1 across several chordate species. Genome or scaffold position is indicated above each HotairM1 locus.
Figure 3. In situ hybridization (ish) in B. lanceolatum and X. tropicallis. Anterior to the left, dorsal is up. (A,A’) Fluorescent HCR ish in B. lanceolatum in whole mount for (A) Hox1 and (A’) Hotairm1 in 30 hpf embryos. White arrows mark the anterior and posterior limits of the expression domain. (B,B’) Colorimetric whole mount ish in X. tropicalis tadpoles for (B) hoxa1 and (B’) hotairm1. Black arrows mark the anterior and posterior limits of the expression domain. (C,C’) Fluorescent double HCR ish in B. lanceolatum in whole mount ish of (C) Hox1 and Hotairm1 in a 36 hpf embryo and (C’) the detailed zone where Hotairm1 peaks its expression. Green arrows mark the anterior and posterior limits of Hox1 expression and red arrows mark the ones of Hotairm1.
Figure 4. Isoform switch of hotairm1 expression towards the unspliced state using a morpholino. (A) Detail of the primers (black and red arrowheads) and morpholino (purple box) used for the amplification of the spliced and unspliced isoforms of hotairm1 in X.tropicalis and for the impairment of the splicing in the MO-treated embryos. (B) Expression of hotairm1 across Xenopus developmental stages. (C) Inhibition of the spliced isoform in MO treated embryos of Xenopus at st18. (D) Assessment of the presence of the unspliced isoform of hotairm1 in MO treated embryos as well as in the control embryos at st18.
Figure 5. MO treated embryos and in situ hybridization in MO treated embryos. Anterior to the left, dorsal is up. (A) Control X. tropicalis MO treated embryos with normal development. 60ng hotairm1-MO treated embryos with a posteriorization of the anterior part of the embryo. (B) Whole mount colorimetric ish of otx2 in X. tropicalis stage 26 control embryos and MO treated embryos showing the reduced expression domain of otx2 in MO treated embryos. (C) Whole mount colorimetric ish of engrailed in X. tropicalis stage 26 control embryos and MO treated embryos showing a clear reduction in the expression in the MO treated embryos.
Figure 6. qPCRs between 60 ng MO treated embryos and control samples at stage 18. * shows statistically significance compared with control samples (Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Gapdh was used as a reference gene.
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