We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33 C and 37 C) and reduced at room temperature (22 C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

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A B S T R A C TPurpose RAF inhibitors are effective against melanomas with BRAF V600E mutations but may induce keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cSCCs). The potential of these agents to promote secondary malignancies is concerning. We analyzed cSCC and KA lesions for genetic mutations in an attempt to identify an underlying mechanism for their formation. MethodsFour international centers contributed 237 KA or cSCC tumor samples from patients receiving an RAF inhibitor (either vemurafenib or sorafenib; n ϭ 19) or immunosuppression therapy (n ϭ 53) or tumors that developed spontaneously (n ϭ 165). Each sample was profiled for 396 known somatic mutations across 33 cancer-related genes by using a mass spectrometric-based genotyping platform. ResultsMutations were detected in 16% of tumors (38 of 237), with five tumors harboring two mutations. Mutations in TP53, CDKN2A, HRAS, KRAS, and PIK3CA were previously described in squamous cell tumors. Mutations in MYC, FGFR3, and VHL were identified for the first time. A higher frequency of activating RAS mutations was found in tumors from patients treated with an RAF inhibitor versus populations treated with a non-RAF inhibitor (21.1% v 3.2%; P Ͻ .01), although overall mutation rates between treatment groups were similar (RAF inhibitor, 21.1%; immunosuppression, 18.9%; and spontaneous, 17.6%; P ϭ not significant). Tumor histology (KA v cSCC), tumor site (head and neck v other), patient age (Յ 70 v Ͼ 70 years), and sex had no significant impact on mutation rate or type. ConclusionSquamous cell tumors from patients treated with an RAF inhibitor have a distinct mutational profile that supports a mechanism of therapy-induced tumorigenesis in RAS-primed cells. Conceivably, cotargeting of MEK together with RAF may reduce or prevent formation of these tumors.

Summary The Ten-Eleven-Translocation 2 (TET2) gene, which oxidates 5-methylcytosine in DNA to 5-hydroxylmethylcytosine (5hmC), is a key tumor suppressor frequently mutated in hematopoietic malignancies. However, the molecular regulation of TET2 expression is poorly understood. We show that TET2 is under extensive microRNA (miRNA) regulation and such TET2-targeting is an important pathogenic mechanism in hematopoietic malignancies. Using a high-throughput 3′UTR activity screen, we identify >30 miRNAs that inhibit TET2 expression and cellular 5hmC. Forced expression of TET2-targeting miRNAs in vivo disrupts normal hematopoiesis, leading to hematopoietic expansion and/or myeloid differentiation bias, whereas co-expression of TET2 corrects these phenotypes. Importantly, several TET2-targeting miRNAs, including miR-125b, miR-29b, miR-29c, miR-101, and miR-7, are preferentially overexpressed in TET2-wildtype acute myeloid leukemia. Our results demonstrate the extensive roles of miRNAs in functionally regulating TET2 and cellular 5hmC, and reveal miRNAs with previously unrecognized oncogenic potential. Our work suggests that TET2-targeting miRNAs might be exploited in cancer diagnosis.