Conservation of a core neurite transcriptome across neuronal types and species

The intracellular localization of mRNAs allows neurons to control gene expression in neurite extensions (axons and dendrites) and respond rapidly to local stimuli. This plays an important role in diverse processes including neuronal growth and synaptic plasticity, which in turn serves as a foundation for learning and memory. Recent high-throughput analyses have revealed that neurites contain hundreds to thousands of mRNAs, but an analysis comparing the transcriptomes derived from these studies has been lacking. Here we analyze 20 datasets pertaining to neuronal mRNA localization across species and neuronal types and identify a conserved set of mRNAs that had robustly localized to neurites in a high number of the studies. The set includes mRNAs encoding for ribosomal proteins and other components of the translation machinery, mitochondrial proteins, cytoskeletal components, and proteins associated with neurite formation. Our combinatorial analysis provides a unique resource for future hypothesis-driven research. This article is categorized under: RNA Export and Localization > RNA Localization RNA Evolution and Genomics > Computational Analyses of RNA RNA Methods > RNA Analyses in Cells

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the anti-sense oligo (ASO) to efficiently and specifically downregulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing

The parasitic lifestyle of an archaeal symbiont

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology

Massively parallel identification of mRNA localization elements in primary cortical neurons

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called ‘zipcodes’. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3′ untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons—cell bodies and neurites—with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes

P2Y receptors and pain transmission

It is widely accepted that the most important ATP receptors involved in pain transmission belong to the P2X3 and P2X2/3 subtypes, selectively expressed in small diameter dorsal root ganglion (DRG) neurons. However, several types of the metabotropic ATP (P2Y) receptors have also been found in primary afferent neurons; P2Y1 and P2Y2 receptors are typically expressed in small, nociceptive cells. Here we review the results available on the involvement of P2Y receptors in the modulation of pain transmission

The P2 Receptor Antagonist PPADS Supports Recovery from Experimental Stroke In Vivo

BACKGROUND: After ischemia of the CNS, extracellular adenosine 5'-triphosphate (ATP) can reach high concentrations due to cell damage and subsequent increase of membrane permeability. ATP may cause cellular degeneration and death, mediated by P2X and P2Y receptors. METHODOLOGY/PRINCIPAL FINDINGS: The effects of inhibition of P2 receptors by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on electrophysiological, functional and morphological alterations in an ischemia model with permanent middle cerebral artery occlusion (MCAO) were investigated up to day 28. Spontaneously hypertensive rats received PPADS or vehicle intracerebroventricularly 15 minutes prior MCAO for up to 7 days. The functional recovery monitored by qEEG was improved by PPADS indicated by an accelerated recovery of ischemia-induced qEEG changes in the delta and alpha frequency bands along with a faster and sustained recovery of motor impairments. Whereas the functional improvements by PPADS were persistent at day 28, the infarct volume measured by magnetic resonance imaging and the amount of TUNEL-positive cells were significantly reduced by PPADS only until day 7. Further, by immunohistochemistry and confocal laser scanning microscopy, we identified both neurons and astrocytes as TUNEL-positive after MCAO. CONCLUSION: The persistent beneficial effect of PPADS on the functional parameters without differences in the late (day 28) infarct size and apoptosis suggests that the early inhibition of P2 receptors might be favourable for the maintenance or early reconstruction of neuronal connectivity in the periinfarct area after ischemic incidents

ATP-Evoked Intracellular Ca Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea

Hearing and its protection is regulated by ATP-evoked Ca2+ signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca2+ imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca2+ signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca2+]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca2+ transients in all three cell types, showing desensitization. Inhibiting the Ca2+ signaling of the ionotropic P2X (omission of extracellular Ca2+) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca2+ stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca2+-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca2+ leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca2+ signaling in these cells. Differences in Ca2+ homeostasis and ATP-induced Ca2+ signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology

Architecture of the biofilm-associated archaic CupE pilus from Pseudomonas aeruginosa

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation in P. aeruginosa

Role of the purinergic and noradrenergic components in the potentiation by endothelin-1 of the sympathetic contraction of the rabbit central ear artery during cooling

1. To examine the role of the purinergic and noradrenergic components in the potentiation of endothelin-1 on the vascular response to sympathetic nerve stimulation, we recorded the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz) under different conditions, at 37°C and during cooling (30°C). 2. Electrical field stimulation produced frequency-dependent contraction, which was reduced during cooling (about 60% for 8 Hz). Both at 37°C and 30°C, phentolamine (1 μM) or blockade of α(1)-adrenoceptors with prazosin (1 μM) reduced, whereas blockade of α(2)-adrenoceptors with yohimbine (1 μM) increased, the contraction to electrical field stimulation. This contraction was increased after desensitization of P2-receptors with α,β-methylene adenosine 5′-triphosphate (α,β-meATP, 3 μM) at 37°C but not at 30°C, and was not modified by blockade of P2-receptors with pyridoxalphosphate-6-azophenyl-2,4′-disulphonic acid (PPADS, 30 μM) at either temperature. 3. Endothelin-1 (1, 3 and 10 nM) at 37°C did not affect, but at 30°C it potentiated in a concentration-dependent manner the contraction to electrical field stimulation (from 28±6 to 134±22%, for 8 Hz). At 37°C, endothelin-1 in the presence of phentolamine or prazosin, but not in that of yohimbine, α,β-meATP or PPADS, potentiated the contraction to electrical stimulation. At 30°C, phentolamine or yohimbine reduced, prazosin or PPADS did not modify and α,β-meATP slightly increased the potentiation by endothelin-1 of the response to electrical stimulation. 4. The arterial contraction to ATP (2 mM) and the α(2)-adrenoceptor agonist BHT-920 (10 μM), but not that to (−)-noradrenaline (1 μM), was potentiated by endothelin-1 at both 37°C and 30°C. 5. These results in the rabbit central ear artery suggest that the sympathetic response: (a) at 37°C, could be mediated mainly by activation of α(1)-adrenoceptors, with low participation of P2-receptors, (b) is diminished during cooling, probably by a reduction in the participation of α(1)-adrenoceptors, and in this condition the response could be mediated in part by P2-receptors, and (c) is potentiated by endothelin-1 during cooling, probably by increasing the response of both postjunctional α(2)-adrenoceptors and P2-receptors