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.

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 the 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 antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral 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.

Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains.

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

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.

Identification of sortase substrates by specificity profiling.

Sortases catalyze the attachment of surface proteins to the peptidoglycan layer of gram-positive bacteria and further represent powerful tools of protein chemistry. During catalysis sortases cleave a donor substrate containing the LPxTG (x=any amino acid) sorting motif under formation of an enzyme-bound thioester and ligate this intermediate to an acceptor protein containing an N-terminal glycine residue. In addition to the well-established sortase A of Staphylococcus aureus several homologs of this enzyme have been identified in the genomes of gram-positive bacteria. We have profiled the specificity of seven sortases of Staphylococci and Streptococci origin and observed that sortases of the latter class displayed a more relaxed specificity for donor and acceptor substrates than their Staphylococci counterparts. Streptococci sortases prefer an LPKLG donor substrate sequence compared to the canonical sorting motif LPKTG. These findings might facilitate the use of Streptococci sortases as tools of protein chemistry.