LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 (gib005Δ8/+)) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability

Targeted RNA knockdown by a type III CRISPR-Cas complex in zebrafish

RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects