Primary cilia are microtubule-based cell organelles important for cellular communication. Since they are involved in the regulation of numerous signalling pathways, defects in cilia development or function are associated with genetic disorders, collectively called ciliopathies. Besides their ciliary functions, recent research has shown that several ciliary proteins are involved in the coordination of the actin cytoskeleton. Although ciliary and actin phenotypes are related, the exact nature of their interconnection remains incompletely understood. Here, we show that the protein BBS6, associated with the ciliopathy Bardet-Biedl syndrome, cooperates with the actin-bundling protein Fascin-1 in regulating filopodia and ciliary signalling. We found that loss of Bbs6 affects filopodia length potentially via attenuated interaction with Fascin-1. Conversely, loss of Fascin-1 leads to a ciliary phenotype, subsequently affecting ciliary Wnt signalling, possibly in collaboration with BBS6. Our data shed light on how ciliary proteins are involved in actin regulations and provide new insight into the involvement of the actin regulator Fascin-1 in ciliogenesis and cilia-associated signalling. Advancing our knowledge of the complex regulations between primary cilia and actin dynamics is important to understand the pathogenic consequences of ciliopathies.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

Interleukin-1β (IL-1β)-induced signaling is one of the most important pathways in regulating inflammation and immunity. The assembly of the receptor complex, consisting of the ligand IL-1β, the IL-1 receptor (IL-1R) type 1 (IL1R1), and the IL-1R accessory protein (IL1RAP), initiates this signaling. However, how the IL1R1-associated complex is regulated remains elusive. Angiopoietin like 3 (ANGPTL3), a key inhibitor of plasma triglyceride clearance, is mainly expressed in the liver and exists in both intracellular and extracellular secreted forms. Presently, ANGPTL3 has emerged as a highly promising drug target for hypertriglyceridemia and associated cardiovascular diseases. However, most studies have focused on the secreted form of ANGPTL3, while its intracellular role is still largely unknown. Here, we report that intracellular ANGPTL3 acts as a negative regulator of IL-1β-triggered signaling. Overexpression of ANGPTL3 inhibited IL-1β-induced NF-κB activation and the transcription of inflammatory genes in HepG2, THP1, and HEK293T cells, while knockdown or knockout of ANGPTL3 resulted in opposite effects. Mechanistically, ANGPTL3 interacted with IL1R1 and IL1RAP through its intracellular C-terminal fibrinogen-like domain (FLD) and disrupted the assembly of the IL1R1-associated complex. Taken together, our study reveals a novel role for ANGPTL3 in inflammation, whereby it inhibits the physiological interaction between IL1R1 and IL1RAP to maintain immune tolerance and homeostasis in the liver.

Endogenous retroviruses (ERVs) are an important component of the transposable elements, which constitute ∼50% of the mammalian genome. They exhibit dynamic expression during early embryonic development and are engaged in numerous biological processes. Therefore, their expression must be closely monitored by cells. Studies on the expression regulation of ERVs have been focused on mouse embryonic stem cells (ESCs) and early embryonic development. This review encapsulates recent advances in the modulation of ERVs in mouse ESCs and preimplantation embryos. The review first touches on the classification, expression, and functions of ERVs in mouse ESCs and early embryos. Next, the review mainly discusses the ERV modulation strategies from the perspectives of transcription, epigenetic modification, nucleosome/chromatin assembly, and post-transcriptional control.

The effective proliferation and differentiation of trophoblast stem cells (TSCs) is indispensable for the development of the placenta, which is the key to maintaining normal fetal growth during pregnancy. Kruppel-like factor 5 (Klf5) is implicated in the activation of pluripotent gene expression in embryonic stem cells (ESCs), yet its function in TSCs is poorly understood. Here, we show that Klf5 knockdown resulted in downregulation of core TSC-specific genes, consequently causing a rapid differentiation of TSCs. Consistently, Klf5-depleted embryos lost the ability to establish TSCs in vitro. At the molecular level, Klf5 preferentially occupied the proximal promoter regions and maintained an open chromatin architecture of key TSC-specific genes. Deprivation of Klf5 impaired the enrichment of p300, a major histone acetyl transferase of H3 lysine 27 acetylation (H3K27ac), and further decreased the occupancy of H3K27ac on promoters, leading to decreased transcriptional activity of TSC pluripotency-related genes. Thus, our findings highlight a novel mechanism of Klf5 in regulating the self-renewal and differentiation of TSCs, and provide a reference for understanding placental development and improving pregnancy rates.

Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to contribute to rescue such mutations. A number of these were based on the capacity of human mitochondria to import RNAs from the cytosol and were designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of the Brome mosaic virus or the Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy opens a powerful approach to eliminate mutant organellar transcripts and to analyze the control and communication of the human organellar genetic system.

Microtubule networks support many cellular processes and have a highly ordered architecture. However, due to the limited axial resolution of conventional light microscopy, the structural features of these networks cannot be resolved in three-dimensional (3D) space. Here, we use customized ultra-high resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells. We find that the microtubule minus-ends associated protein CAMSAPs localize at a portion of microtubule intersections. Further investigation shows that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance. We find that CAMSAPs recognize microtubule defects, which are often associated with microtubule intersections, and then recruit katanin to remove the damaged microtubules. Therefore, the CAMSAP-katanin complex is a regulating module for the distance between microtubules. Taken together, our results characterize the architecture of the cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.

The super elongation complex (SEC) containing P-TEFb plays a critical role in regulating transcription elongation. AFF1 and AFF4, members of the AF4/FMR2 family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. We here reveal differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSSs, while AFF4 is enriched downstream of the TSSs. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4 bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate Pol II transcription.