A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Organismal landscape of clock cells and circadian gene expression in Drosophila.

Background: Circadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in Drosophila some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF). Despite these findings and the ample knowledge of the molecular clockwork, it is still unknown how circadian gene expression in Drosophila is achieved across the body. Results: Here, we used single-cell and bulk RNAseq data to identify cells within the fly that express core-clock components. Surprisingly, we found that less than a third of the cell types in the fly express core-clock genes. Moreover, we identified Lamina wild field (Lawf) and Ponx-neuro positive (Poxn) neurons as putative new circadian neurons. In addition, we found several cell types that do not express core clock components but are highly enriched for cyclically expressed mRNAs. Strikingly, these cell types express the PDF receptor (Pdfr), suggesting that PDF drives rhythmic gene expression in many cell types in flies. Other cell types express both core circadian clock components and Pdfr, suggesting that in these cells, PDF regulates the phase of rhythmic gene expression. Conclusions: Together, our data suggest three different mechanisms generate cyclic daily gene expression in cells and tissues: canonical endogenous canonical molecular clock, PDF signaling-driven expression, or a combination of both.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome.

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1,000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome.

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in the presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

Viruses are parasites, relying on the cells they infect to make more of themselves. In doing so they change how an infected cell turns its genes on and off, forcing it to build new virus particles and turning off the immune surveillance that would allow the body to intervene. This is how SARS-CoV-2, the virus that causes COVID, survives with a genome that carries instructions to make just 29 proteins. One of these proteins, known as Nsp14, is involved in both virus reproduction and immune escape. Previous work has shown that it interacts with IMPDH2, the cellular enzyme that controls the production of the building blocks of the genetic code. The impact of this interaction is not clear. To find out more, Zaffagni et al. introduced 26 of the SARS-CoV-2 proteins into human cells one at a time. Nsp14 had the most dramatic effect, dialing around 4,000 genes up or down and changing how the cell interprets over 1,000 genes. Despite being just one protein, it mimicked the genetic changes seen during real SARS-CoV-2 infection. Blocking IMPDH2 partially reversed the effects, which suggests that the interaction of Nsp14 with the enzyme might be responsible for the effects of SARS-CoV-2 on the genes of the cell. Understanding how viral proteins affect cells can explain what happens during infection. This could lead to the discovery of new treatments designed to counteract the effects of the virus. Further work could investigate whether interfering with Nsp14 helps cells to overcome infection.

Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor based on standard iGEM parts.

The diversity and flexibility of life offers a wide variety of molecules and systems useful for biosensing. A biosensor device should be robust, specific and reliable. Inorganic arsenic is a highly toxic water contaminant with worldwide distribution that poses a threat to public health. With the goal of developing an arsenic biosensor, we designed an incoherent feed-forward loop (I-FFL) genetic circuit to correlate its output pulse with the input signal in a relatively time-independent manner. The system was conceived exclusively based on the available BioBricks in the iGEM Registry of Standard Biological Parts. The expected behavior in silico was achieved; upon arsenic addition, the system generates a short-delayed reporter protein pulse that is dose dependent to the contaminant levels. This work is an example of the power and variety of the iGEM Registry of Standard Biological Parts, which can be reused in different sophisticated system designs like I-FFLs. Besides the scientific results, one of the main impacts of this synthetic biology project is the influence it had on team's members training and career choices which are summarized at the end of this article.