Tumor protein Tctp regulates axon development in the embryonic visual system.

The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.

Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes.

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

Localised dynactin protects growing microtubules to deliver oskar mRNA to the posterior cortex of the Drosophila oocyte.

The localisation of oskar mRNA to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Kinesin 1 transports oskar mRNA to the oocyte posterior along a polarised microtubule cytoskeleton that grows from non-centrosomal microtubule organising centres (ncMTOCs) along the anterior/lateral cortex. Here, we show that the formation of this polarised microtubule network also requires the posterior regulation of microtubule growth. A missense mutation in the dynactin Arp1 subunit causes most oskar mRNA to localise in the posterior cytoplasm rather than cortically. oskar mRNA transport and anchoring are normal in this mutant, but the microtubules fail to reach the posterior pole. Thus, dynactin acts as an anti-catastrophe factor that extends microtubule growth posteriorly. Kinesin 1 transports dynactin to the oocyte posterior, creating a positive feedback loop that increases the length and persistence of the posterior microtubules that deliver oskar mRNA to the cortex.

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity.

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions.