Divergent lncRNAs that are transcribed in the opposite direction to nearby protein-coding genes comprise a significant proportion (∼20%) of total lncRNAs in mammalian genomes. Through genome-wide analysis, we found that the distribution of this lncRNA class strongly correlates with essential developmental regulatory genes. In pluripotent cells, divergent lncRNAs regulate the transcription of nearby genes. As an example, the divergent lncRNA Evx1as promotes transcription of its neighbor gene, EVX1, and regulates mesendodermal differentiation. At a single-cell level, early broad expression of Evx1as is followed by a rapid, high-level transcription of EVX1, supporting the idea that Evx1as plays an upstream role to facilitate EVX1 transcription. Mechanistically, Evx1as RNA binds to regulatory sites on chromatin, promotes an active chromatin state, and interacts with Mediator. Based on our analyses, we propose that the biological function of thousands of uncharacterized lncRNAs of this class may be inferred from the role of their neighboring adjacent genes.

A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZβ, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.