A virus‐derived microRNA targets immune response genes during SARS‐CoV‐2 infection

SARS‐CoV‐2 infection results in impaired interferon response in patients with severe COVID‐19. However, how SARS‐CoV‐2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS‐CoV‐2‐infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus‐derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3′UTR of interferon‐stimulated genes and represses their expression in a miRNA‐like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID‐19 patients. We propose that SARS‐CoV‐2 can potentially employ a virus‐derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon‐mediated immune response.     

Aging‐related cell type‐specific pathophysiologic immune responses that exacerbate disease severity in aged COVID‐19 patients

Coronavirus disease 2019 (COVID‐19) is especially severe in aged patients, defined as 65 years or older, for reasons that are currently unknown. To investigate the underlying basis for this vulnerability, we performed multimodal data analyses on immunity, inflammation, and COVID‐19 incidence and severity as a function of age. Our analysis leveraged age‐specific COVID‐19 mortality and laboratory testing from a large COVID‐19 registry, along with epidemiological data of ~3.4 million individuals, large‐scale deep immune cell profiling data, and single‐cell RNA‐sequencing data from aged COVID‐19 patients across diverse populations. We found that decreased lymphocyte count and elevated inflammatory markers (C‐reactive protein, D‐dimer, and neutrophil–lymphocyte ratio) are significantly associated with age‐specific COVID‐19 severities. We identified the reduced abundance of naïve CD8 T cells with decreased expression of antiviral defense genes (i.e., IFITM3 and TRIM22) in aged severe COVID‐19 patients. Older individuals with severe COVID‐19 displayed type I and II interferon deficiencies, which is correlated with SARS‐CoV‐2 viral load. Elevated expression of SARS‐CoV‐2 entry factors and reduced expression of antiviral defense genes (LY6E and IFNAR1) in the secretory cells are associated with critical COVID‐19 in aged individuals. Mechanistically, we identified strong TGF‐beta‐mediated immune–epithelial cell interactions (i.e., secretory‐non‐resident macrophages) in aged individuals with critical COVID‐19. Taken together, our findings point to immuno‐inflammatory factors that could be targeted therapeutically to reduce morbidity and mortality in aged COVID‐19 patients.     

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal‐to‐zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc‐miR‐205 and bovine bta‐miR‐10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc‐miR‐205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte‐to‐embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.     

Tissue and Process Specific microRNA–mRNA Co-Expression in Mammalian Development and Malignancy

An association between enrichment and depletion of microRNA (miRNA) binding sites, 3′ UTR length, and mRNA expression has been demonstrated in various developing tissues and tissues from different mature organs; but functional, context-dependent miRNA regulations have yet to be elucidated. Towards that goal, we examined miRNA–mRNA interactions by measuring miRNA and mRNA in the same tissue during development and also in malignant conditions. We identified significant miRNA-mediated biological process categories in developing mouse cerebellum and lung using non-targeted mRNA expression as the negative control. Although miRNAs in general suppress target mRNA messages, many predicted miRNA targets demonstrate a significantly higher level of co-expression than non-target genes in developing cerebellum. This phenomenon is tissue specific since it is not observed in developing lungs. Comparison of mouse cerebellar development and medulloblastoma demonstrates a shared miRNA–mRNA co-expression program for brain-specific neurologic processes such as synaptic transmission and exocytosis, in which miRNA target expression increases with the accumulation of multiple miRNAs in developing cerebellum and decreases with the loss of these miRNAs in brain tumors. These findings demonstrate the context-dependence of miRNA–mRNA co-expression.     

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult‐type granulosa cell tumors (AGCTs), introduces a target site for miR‐1236, which causes haploinsufficiency of the tumor‐suppressor FOXL2. This miR‐1236‐mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA‐loaded RNA‐induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR‐1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation‐mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA‐targeting disease‐associated mutations in the CDS by forming a non‐canonical miRISC.     

Analysis and pharmacological modulation of senescence in human epithelial stem cells

Human epithelial stem cells (ESCs) are characterized by long‐term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly‐ectodermal dysplasia‐clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ‐secretase inhibitor; N‐[N‐(3, 5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine T‐butyl ester). All cells were able to differentiate in vitro but exhibited variable self‐renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up‐ or down‐regulated during their lifetime, thus allowing to identify druggable gene networks and off‐the‐shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.     

HDAC6 inhibition restores TDP‐43 pathology and axonal transport defects in human motor neurons with TARDBP mutations

TDP‐43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP‐43, are one of the common causes of familial ALS. In this study, we investigate TDP‐43 protein behavior in induced pluripotent stem cell (iPSC)‐derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP‐43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP‐43, C‐terminal fragments, and phospho‐TDP‐43. By generating iPSC lines with allele‐specific tagging of TDP‐43, we find that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP‐43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP‐43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP‐43 pathology.     

METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.