The role of midbody-associated mRNAs in regulating abscission.

Midbodies function during telophase to regulate the abscission step of cytokinesis. Until recently, it was thought that abscission-regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting the MB. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. We propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

Mechanical Fractionation of Cultured Neuronal Cells into Cell Body and Neurite Fractions.

Many cells contain spatially defined subcellular regions that perform specialized tasks enabled by localized proteins. The subcellular distribution of these localized proteins is often facilitated by the subcellular localization of the RNA molecules that encode them. A key question in the study of this process of RNA localization is the characterization of the transcripts present at a given subcellular location. Historically, experiments aimed at answering this question have centered upon microscopy-based techniques that target one or a few transcripts at a time. However, more recently, the advent of high-throughput RNA sequencing has allowed the transcriptome-wide profiling of the RNA content of subcellular fractions. Here, we present a protocol for the isolation of cell body and neurite fractions from neuronal cells using mechanical fractionation and characterization of their RNA content. Graphic abstract: Fractionation of neuronal cells and analysis of subcellular RNA contents.

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons.

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation.