SUMMARY Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs—a long ncRNA, a circular RNA, and two microRNAs—using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7–targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread post-transcriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. Here we found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings suggested and supported a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin–proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function, KRIT1, CCM2 and PDCD10. Here we show that the HEG1 receptor, linked to CCM genes in zebrafish, is selectively expressed in endothelial cells and that Heg1-/- mice exhibit defective integrity of the heart, blood vessels and lymphatic vessels. In contrast, Heg1-/-;Ccm2+/lacZ and Ccm2lacZ/lacZ mice die early in development due to a failure of nascent endothelial cells to associate into patent vessels, a phenotype shared by deficient zebrafish embryos and reproduced by deficient endothelial cells ex vivo. These cardiovascular defects are associated with abnormal endothelial junctions like those observed in human CCMs, and biochemical and cellular imaging studies identify a cell autonomous pathway in which HEG1 receptors couple to KRIT1 at cell junctions. These studies identify HEG1-CCM signaling as a critical regulator of cardiovascular organ formation and integrity.

Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding

SUMMARYNoncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs-a long ncRNA, a circular RNA, and two microRNAs-using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more efficient than previously described examples of target-directed microRNA degradation, which come from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables the accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network. HIGHLIGHTSA long noncoding RNA, a circular RNA, and two microRNAs form a regulatory network The Cyrano long noncoding RNA directs potent, multiple-turnover destruction of miR-7

We have defined inactive ␣ and fragments of ␤-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the ␣ fragment increased complemented enzyme activity by up to 4 orders of magnitude. ␤-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date. P rotein-protein interactions are involved in every cellular process ranging from gene expression and signal transduction to cell division and differentiation, yet they have been among the most difficult aspects of cell biology to study. Standard biochemical methods have yielded most of the available information about such interactions, but these assays often are limited by the available reagents such as monoclonal antibodies for immunoprecipitation or lack the appropriate cellular context.The development of fusion protein-based assays such as the yeast two-hybrid method (1) has expanded the potential for studying protein interactions in intact cells greatly. However, this assay relies on the transcription of a reporter gene; consequently it is not applicable to studies of the kinetics of protein-protein interactions and is unable to detect the interaction of compartmentalized proteins such as receptors at the cell surface. A method based on fluorescence resonance energy transfer provided a further advance and currently is one of the most accurate methods used to monitor dynamic interactions (2). However, the incremental changes in fluorescence assayed by fluorescence resonance energy transfer are small, and the stringent steric requirements for detecting the interacting proteins can restrict the utility of this technique.Assays based on the complementation of enzyme fragments fused to interacting proteins that regenerate enzymatic activity after dimerization are particularly well suited for monitoring inducible protein interactions (reviewed in ref.3). These systems have important advantages including low-level expression of the test proteins, generation of signal as a direct result of the interaction, and enzymatic amplification. As a result, they are highly sensitive and physiologically relevant assays (4). Additionally, assays based on enzyme complementation can be performed in any cell type of interest or in diverse cellular compartments such as the nucleus, secretory vesicles, or plasma membrane.The class A ␤-lactamases are particularly attractive candidates for an assay based on enzyme fragment complementation because of the fact that they are monomeric and of relatively small si...

SUMMARY Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralogue, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development as well as postnatal vessel growth.

Background and Purpose The Heart of Glass (HEG) receptor binds KRIT1 and functions with KRIT1, CCM2 and PDCD10 in a common signaling pathway required for heart and vascular development. Mutations in KRIT1, CCM2 and PDCD10 also underlie human cerebral cavernous malformation (CCM), and postnatal loss of these genes in the mouse endothelium results in rapid CCM formation. Here we test the role of HEG in CCM formation in mice and humans. Methods We constitutively or conditionally deleted Heg and/or Ccm2 genes in genetically modified mice. Mouse embryos, brain and retina tissues were analyzed to assess CCM lesion formation. Results CCMs form in postnatal mice with Ccm2−/− but not Heg−/− or Heg−/−;Ccm2+/−endothelial cells. Consistent with these findings, human patients with CCM who lack exonic mutations in KRIT1, CCM2 or PDCD10 do not have mutations in HEG. Conclusion These findings suggest that the HEG-CCM signaling functions during cardiovascular development and growth, while CCMs arise due to loss of HEG-independent CCM signaling in the endothelium of the central nervous system after birth.