mRNA stability and m6A are major determinants of subcellular mRNA localization in neurons.

For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here, we assess the role of mRNA stability in localization by combining the isolation of the soma and neurites of mouse primary cortical and mESC-derived neurons, SLAM-seq, m6A-RIP-seq, the perturbation of mRNA destabilization mechanisms, and the analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m6A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons.

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.