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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

Circular RNAs (circRNAs) are a subclass of noncoding RNAs widely expressed in mammalian cells. We report here the tumorigenic capacity of a circRNA derived from angiomotin-like1 (circ-Amotl1). Circ-Amotl1 is highly expressed in patient tumor samples and cancer cell lines. Single-cell inoculations using circ-Amotl1-transfected tumor cells showed a 30-fold increase in proliferative capacity relative to control. Agarose colony-formation assays similarly revealed a 142-fold increase. Tumor-take rate in nude mouse xenografts using 6-day (219 cells) and 3-day (9 cells) colonies were 100%, suggesting tumor-forming potential of every cell. Subcutaneous single-cell injections led to the formation of palpable tumors in 41% of mice, with tumor sizes >1 cm 3 in 1 month. We further found that this potent tumorigenicity was triggered through interactions between circ-Amotl1 and c-myc. A putative binding site was identified in silico and tested experimentally. Ectopic expression of circ-Amotl1 increased retention of nuclear c-myc, appearing to promote c-myc stability and upregulate c-myc targets. Expression of circ-Amotl1 also increased the affinity of c-myc binding to a number of promoters. Our study therefore reveals a novel function of circRNAs in tumorigenesis, and this subclass of noncoding RNAs may represent a potential target in cancer therapy.

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell–derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

The hexanucleotide repeat expansion (HRE) GGGGCC (G 4 C 2 ) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G 4 C 2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G 4 C 2 repeats identified RanGAP ( Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention. A candidate-based genetic screen in Drosophila expressing 30 G 4 C 2 -repeat-containing RNAs finds that RanGAP, a key regulator of nucleocytoplasmic transport, is a potent suppressor of neurodegeneration; the defects caused by the G 4 C 2 repeat expansions can be rescued with antisense oligonucleotides or small molecules targeting the G-quadruplexes. A novel mechanism of neurodegeneration The most common cause of the debilitating disease amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion GGGGCC (G4C2) in the C9orf72 gene. Two studies in this issue use contrasting methods to arrive at a molecular mechanism that may cause a familial form of the disease. Using a candidate-based genetic screen in Drosophila expressing 30 G 4 C 2 repeats (Ke Zhang et al .) or an unbiased genetic screen in Drosophila expressing 8, 28 or 58 G 4 C 2 repeat-containing transcripts (Brian Freibaum et al .), the two groups sought genes that enhance or suppress the disease phenotype. Zhang et al . identify the gene encoding RanGAP, a key regulator of nucleocytoplasmic transport, and Freibaum et al . identifies genes that encode components of the nuclear pore and the nucleocytoplasmic transport machinery. Both papers show deficits in nucleocytoplasmic transport in Drosophila cells expressing G 4 C 2 repeats and in iPSC-derived neurons from ALS patients. Zhang et al . show that these defects can be rescued with antisense oligonucleotides or small molecules targeting the G-quadruplexes.

Key Points MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Biogenesis of miRNA is under tight temporal and spatial control. Dysregulation of miRNA is associated with many human diseases, particularly cancer and neurodevelopmental disorders. Regulation takes place at multiple levels including transcription, Drosha processing, Dicer processing, RNA editing, RNA methylation, uridylation, adenylation, Argonaute modification and RNA decay. This Review summarizes our current understanding of how miRNAs are made and regulated, with a focus on animal systems. In animals, microRNAs (miRNAs) are ∼22 nucleotides in length and are produced by two RNase III proteins — Drosha and Dicer. Their biogenesis is regulated at multiple levels, including at the level of miRNA transcription; by Drosha and Dicer processing; by their modification through RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and by RNA decay. MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins — Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.

An unbiased genetic screen in Drosophila expressing G 4 C 2 -repeat-containing transcripts (repeats that in human cause pathogenesis in C9orf72 -related neurological disease) finds genes that encode components of the nuclear pore and nucleocytoplasmic transport machinery, and reveals that G 4 C 2 expanded-repeat-induced alterations in nucleocytoplasmic transport contribute to C9orf72 pathology and neurodegeneration. A novel mechanism of neurodegeneration The most common cause of the debilitating disease amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion GGGGCC (G4C2) in the C9orf72 gene. Two studies in this issue use contrasting methods to arrive at a molecular mechanism that may cause a familial form of the disease. Using a candidate-based genetic screen in Drosophila expressing 30 G 4 C 2 repeats (Ke Zhang et al .) or an unbiased genetic screen in Drosophila expressing 8, 28 or 58 G 4 C 2 repeat-containing transcripts (Brian Freibaum et al .), the two groups sought genes that enhance or suppress the disease phenotype. Zhang et al . identify the gene encoding RanGAP, a key regulator of nucleocytoplasmic transport, and Freibaum et al . identifies genes that encode components of the nuclear pore and the nucleocytoplasmic transport machinery. Both papers show deficits in nucleocytoplasmic transport in Drosophila cells expressing G 4 C 2 repeats and in iPSC-derived neurons from ALS patients. Zhang et al . show that these defects can be rescued with antisense oligonucleotides or small molecules targeting the G-quadruplexes. The GGGGCC (G 4 C 2 ) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia 1 , 2 . The basis for pathogenesis is unknown. To elucidate the consequences of G 4 C 2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G 4 C 2 -repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72 -related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G 4 C 2 repeats in vitro and in vivo . Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G 4 C 2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72 -related disease. These studies show that a primary consequence of G 4 C 2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus–cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR–Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR–Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2 , modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR–Cas9 screens in defining mechanisms of neurodegenerative diseases. A genome-wide CRISPR screen for suppressors and enhancers of C9ORF72 dipeptide-repeat protein toxicity identifies candidate genes involved in nucleocytoplasmic transport and other pathways including RNA processing and chromatin modification.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20–30 cancer genes 1 – 8 . Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease 9 – 13 than in normal liver 13 – 16 , which enables positive selection to shape the genomic landscape 9 – 13 . Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1 , the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1 S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB , which regulates lipid droplet metabolism in hepatocytes 17 – 19 , and GPAM , which produces storage triacylglycerol from free fatty acids 20 , 21 , also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease. Whole-genome sequencing analysis of somatic mutations in liver samples from patients with chronic liver disease identifies driver mutations in metabolism-related genes such as FOXO1 , and shows that these variants frequently exhibit convergent evolution.

CXC chemokine receptor 4 (CXCR4) has been suggested to play a critical role in cancer metastasis. Some studies have described CXCR4 nuclear localization in metastatic lesions of renal cell carcinoma (RCC), which has been suggested to be correlated with cancer metastasis. However, the underlying mechanism and clinical significance of CXCR4 nuclear localization remains unknown. Here, we show that CXCR4 nuclear localization is more likely to occur in RCC tissues, especially in metastases, and is associated with poor prognosis. CXCR4 nuclear localization requires its nuclear localization sequence (NLS, residues 146-RPRK-149). After the mutation of NLS in CXCR4, CXCR4 nuclear localization in RCC cells is lost. Nuclear localization of CXCR4 promoted RCC tumorigenicity both in vitro and in vivo. Mechanistically, we found that CXCR4 and hypoxia-inducible factor-1α (HIF-1α) colocalized in RCC cells and interacted with each other. Moreover, CXCR4 nuclear localization promoted nuclear accumulation of HIF-1α, thereby promoting the expression of genes downstream of HIF-1α. Reciprocally, nuclear HIF-1α promoted CXCR4 transcription, thus forming a feed-forward loop. Subcellular CXCR4 and HIF-1α expression levels were independent adverse prognostic factors and could be combined with TNM stage to generate a predictive nomogram of the clinical outcome of patients with RCC. Therefore, our findings indicate that CXCR4 nuclear translocation plays a critical role in RCC metastasis and may serve as a prognostic biomarker and potential therapeutic target.