Rapid nuclear deadenylation of mammalian messenger RNA

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization

Quantitative X-ray wavefront measurements of Fresnel zone plate and K-B mirrors using phase retrieval

A scanning coherent diffraction imaging method was used to reconstruct the X-ray wavefronts produced by a Fresnel zone plate (FZP) and by Kirkpatrick-Baez (KB) focusing mirrors. The ptychographical measurement was conducted repeatedly by placing a lithographed test sample at different defocused planes. The wavefronts, recovered by phase-retrieval at well-separated planes, show good consistency with numerical propagation results, which provides a self-verification. The validity of the obtained FZP wavefront was further confirmed with theoretical predictions

Sites of transcription initiation drive mRNA isoform selection

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity

Combined X-ray microfluorescence and atomic force microscopy studies of Mg distribution in whole cells

We present in this paper a novel methodology that combines scanning x\u2010ray fluorescencee microscopy and atomic force microscopy. The combination of these two techniques allows the determination of a concentration map of Mg in whole (not sectioned) cells

Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-κB (NF-κB). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection

Kinetic modelling of competition and depletion of shared miRNAs by competing endogenous RNAs

Non-conding RNAs play a key role in the post-transcriptional regulation of mRNA translation and turnover in eukaryotes. miRNAs, in particular, interact with their target RNAs through protein-mediated, sequence-specific binding, giving rise to extended and highly heterogeneous miRNA-RNA interaction networks. Within such networks, competition to bind miRNAs can generate an effective positive coupling between their targets. Competing endogenous RNAs (ceRNAs) can in turn regulate each other through miRNA-mediated crosstalk. Albeit potentially weak, ceRNA interactions can occur both dynamically, affecting e.g. the regulatory clock, and at stationarity, in which case ceRNA networks as a whole can be implicated in the composition of the cell's proteome. Many features of ceRNA interactions, including the conditions under which they become significant, can be unraveled by mathematical and in silico models. We review the understanding of the ceRNA effect obtained within such frameworks, focusing on the methods employed to quantify it, its role in the processing of gene expression noise, and how network topology can determine its reach.Comment: review article, 29 pages, 7 figure

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies

MicroRNAs are deeply linked to the emergence of the complex octopus brain

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the (Octopus vulgaris). We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains

Neurodegeneration in human brain organoids infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) infection of the nervous system may lead to brain damage, including neurodegeneration. However, lack of suitable experimental models hinders understanding molecular mechanisms and cell-type-specific responses triggered by HSV-1. Here, we infected human brain organoids with HSV-1. Known features of HSV-1 infection such as alteration of neuronal electrophysiology and induction of antisense transcription were confirmed. Full-length mRNA-sequencing revealed aberrant 3’ end formation and poly(A)-tail lengthening. Single-cell RNA-seq and spatial transcriptomics uncovered changes in the cellular composition of the infected organoids caused by viral replication and dysregulation of molecular pathways in cell-type specific manner. Furthermore, hallmarks of early neurodegeneration were observed, namely extracellular matrix disruption, STMN2 and TARDBP/TDP43 downregulation, and upregulation of the AD-related non-coding RNA BC200/BCYRN1. These hallmarks were weaker/absent when infecting with a mutant HSV-1 control. Together, our data indicate that brain organoids serve as a powerful model to study mechanisms of HSV-1-driven neurodegeneration