Depicting lncRNA transcripts targeted by miRNAs.

<p>The figure shows the lncRNA transcripts targeted by miRNAs along with the distribution of Ago types in the targeting complex. The presence and absence of specific Ago type in the RISC complex is represented by the difference in the colors in the heatmap. The targeting miRNA has also been mentioned.</p

Binding alignments of microRNAs targeting predicted zebrafish lncRNA.

<p>The figure shows predicted binding alignment of miR-125a, miR-125b, miR-125c, miR-17a*, miR-20*, miR-210*, miR-29a, miR-29b and miR457a with predicted zebrafish lncRNA.</p

Contribution of each Ago class in targeting lncRNA.

<p>Contribution of each Ago class in targeting lncRNA.</p

A: Complete interaction network of lncRNA, miRNA and mRNA.

<p>The interaction network wherein the experimental miRNA-mRNA interactions are represented in red nodes, the predicted miRNA-lncRNA interactions are represented in blue nodes and total miRNA-mRNA interactions represented as black nodes. <b>B1:</b> An interesting example from the network highlighted in orange showing interactions between of network highlighting <b>miRNA:</b> (a) hsa-mir-9; <b>lncRNA:</b> (d) ENST00000500197.2, (e) ENST00000509783.1, (f) ENST00000511014.1, (h) ENST00000505030.1, (i) ENST00000504246.1; <b>mRNA:</b> (b) ENST00000384838.1, (c) ENST00000262095.2, (g) ENST00000491143.1, (j) ENST00000226574 <b>B2:</b> Another interesting example from the network highlighted in blue showing interactions between <b>miRNA</b>: (1) hsa-miR-196a, (5) hsa-miR-196b*, (13)hsa- miR-196b; <b>lncRNA</b>: (4) ENST00000523790.1; (6) ENST00000489695.1, (12) ENST00000519935.1; <b>mRNA</b>: (2) ENST00000354032.4, (3) ENST00000384852.1, (7) ENST00000313173, (8) ENST0000024215, (9) ENST00000040584, (10) ENST00000304786.7, (11) ENST00000366839.4.</p

The number of reads mapping in each of the individual datasets.

<p>The number of reads mapping in each of the individual datasets.</p

Workflow for the analysis of potential microRNA binding sites on lncRNAs and re-construction of the regulatory interactome.

<p>Workflow for the analysis of potential microRNA binding sites on lncRNAs and re-construction of the regulatory interactome.</p

Reverse Genetics Screen in Zebrafish Identifies a Role of miR-142a-3p in Vascular Development and Integrity

<div><p>MicroRNAs are a well-studied class of non-coding RNA and are known to regulate developmental processes in eukaryotes. Their role in key biological processes such as vasculature development has attracted interest. However, a comprehensive understanding of molecular regulation of angiogenesis and vascular integrity during development remains less explored. Here we identified miRNAs involved in the development and maintenance of vasculature in zebrafish embryos using a reverse genetics approach. Using a combination of bioinformatics predictions and literature based evidences we mined over 701 Human and 329 Zebrafish miRNAs to derive a list of 29 miRNAs targeting vascular specific genes in zebrafish. We shortlisted eight miRNAs and investigated their potential role in regulating vascular development in zebrafish transgenic model. In this screen we identified three miRNAs, namely miR-1, miR-144 and miR-142a-3p that have the potential to influence vascular development in zebrafish. We show that miR-142a-3p mediates vascular integrity and developmental angiogenesis <em>in vivo.</em> Overexpression of miR-142a-3p results in loss of vascular integrity, hemorrhage and vascular remodeling during zebrafish embryonic development, while loss of function of miR-142a-3p causes abnormal vascular remodeling. MiR-142a-3p functions in part by directly repressing <em>cdh5</em> (<em>VE-cadherin).</em> The vascular abnormalities that results from modulation of miR-142a-3p are reminiscent of <em>cdh5</em> perturbation in zebrafish embryos. We also demonstrate that the action of miR-142a on <em>cdh5</em> is potentially regulated by Lmo2, an important transcription factor, known for its role in vasculature development. The miR142a-3p mediated control of <em>cdh5</em> constitutes an additional layer of regulation for maintaining vascular integrity and developmental angiogenesis. These findings have implications in development, wound repair and tumor growth.</p> </div

Overexpression of miR-142a-3p in zebrafish embryos induces vascular defects.

<p>A – D - Confocal images (GFP and GFP/RFP merged) of 10 µM miR-142a-3p duplex injected Tg<i>(fli1:EGFP, gata1a: dsRed)</i> embryos at 2 dpf (Dorsal View of head) depicting dorsal midbrain junction (DMJ) and central arteries (CtA) (10× magnification). A, B - non-injected control embryos and C, D - 10 µM miR-142a-3p duplex injected embryos. A, C – GFP, B, D – GFP/RFP merged. Asterisk sign indicate the site of vascular defect.</p

Overexpression of miRNAs in zebrafish embryos induces specific vascular phenotypes.

<p>A - Schematic of the experimental approach. MiRNA duplexes were ectopically overexpressed in Tg<i>(fli1:EGFP, gata1a: dsRed)</i> zebrafish embryos through microinjection at 1–2 cell stage. The injected embryos were scored at 2 dpf under microscope for visual observation of phenotype under bright field, GFP and RFP filters. Representative images of miRNA-injected Tg<i>(fli1:EGFP, gata1a: dsRed)</i> zebrafish embryos are displayed. B,C,D - Non-injected control (NIC) zebrafish embryos with normal vascular development. E,F,G - Zebrafish embryos injected with miRNA annealing buffer display normal vascular development. H,I,J - Zebrafish embryos injected with miR-144 display reduced or absence of blood in intersegmental vessels. K,L,M - Zebrafish embryos injected with miR-1 display accumulation of blood cells in LDA/YSL. N,O,P - Zebrafish embryos injected with miR-142a-3p display pooling of blood cells in head/trunk region. Q,R,S and T,U,V - Zebrafish embryos injected with miR-181a and miR-181b respectively display no visible phenotype. Zebrafish embryos injected with miR-221, miR-222 and miR-451 display no observable phenotype (figure not shown). Arrowheads indicate the site of vascular defects. The embryos were imaged at 2.5× magnification.</p

Overexpression of miR-142a-3p in zebrafish embryos induces hemorrhage and vascular remodeling.

<p>A - Sequence alignment of mature miR-142a-3p from different species is highlighted with seed sequence in gray color. B – Synteny analysis of human miR-142 on chromosome 17 with zebrafish miR-142a on chromosome 5. Arrow in genes indicates strand information on chromosome. C - Bar graph showing cerebral hemorrhage phenotype in miR-142a-3p duplex injected Tg<i>(fli1:EGFP, gata1a: dsRed)</i> zebrafish embryos at 2 dpf. Data is represented as mean percentage ± SD (standard deviation) collected over 3 independent experiments and n is number embryos analyzed. D - I - Representative image of cerebral hemorrhage phenotype in Tg<i>(fli1:EGFP, gata1a: dsRed)</i> zebrafish embryos at 2 dpf. D,F,H - Non-injected control embryos (NIC) and E,G,I - miR-142a-3p injected embryos. H,I - Embryos stained with <i>o-dianisidine</i>. D–I - 2.5× magnification.</p