RENGE infers gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations.

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation has enabled the inference of gene regulatory networks with causal relationships. However, a snapshot of single-cell CRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a computational method that infers gene regulatory networks using a time-series single-cell CRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its regulatory network. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring gene regulatory networks. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a network consistent with multiple databases and literature. Accurate inference of gene regulatory networks by RENGE would enable the identification of key factors for various biological systems.

IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay.

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1ß- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1ß or TLR stimulus dynamically induced the formation of Regnase-1-ß-transducin repeat-containing protein (ßTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by ßTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-ßTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from ßTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

F-Box Proteins and Cancer.

Controlled protein degradation is essential for the operation of a variety of cellular processes including cell division, growth, and differentiation. Identification of the relations between ubiquitin ligases and their substrates is key to understanding the molecular basis of cancer development and to the discovery of novel targets for cancer therapeutics. F-box proteins function as the substrate recognition subunits of S-phase kinase-associated protein 1 (SKP1)-Cullin1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes. Here, we summarize the roles of specific F-box proteins that have been shown to function as tumor promoters or suppressors. We also highlight proto-oncoproteins that are targeted for ubiquitylation by multiple F-box proteins, and discuss how these F-box proteins are deployed to regulate their cognate substrates in various situations.

Skp2 contributes to cell cycle progression in trophoblast stem cells and to placental development.

All trophoblast subtypes of the placenta are derived from trophoblast stem cells (TSCs). TSCs have the capacity to self-renew, but how the proliferation of these cells is regulated in the undifferentiated state has been largely unclear. We now show that the F-box protein Skp2 regulates the proliferation of TSCs and thereby plays a pivotal role in placental development in mice on the C57BL/6 background. The placenta of Skp2-/- mouse embryos on the C57BL/6 background was smaller than that of their Skp2+/+ littermates, with the mutant embryos also manifesting intrauterine growth retardation. Although the Skp2-/- mice were born alive, most of them died before postnatal day 21, presumably as a result of placental defects. Depletion of Skp2 in TSCs cultured in the undifferentiated state resulted in a reduced rate of proliferation and arrest of the cell cycle in G1 phase, indicative of a defect in self-renewal capacity. The cell cycle arrest apparent in Skp2-deficient TSCs was reversed by additional ablation of the cyclin-dependent kinase inhibitor (CKI) p57 but not by that of the CKI p27. Our results thus suggest that Skp2-mediated degradation of p57 is an important determinant of the self-renewal capacity of TSCs during placental development, at least in mice of certain genetic backgrounds.