Growth cone MKK7 mRNA targeting regulates MAP1b-dependent microtubule bundling to control neurite elongation.

Local mRNA translation in neurons has been mostly studied during axon guidance and synapse formation but not during initial neurite outgrowth. We performed a genome-wide screen for neurite-enriched mRNAs and identified an mRNA that encodes mitogen-activated protein kinase kinase 7 (MKK7), a MAP kinase kinase (MAPKK) for Jun kinase (JNK). We show that MKK7 mRNA localizes to the growth cone where it has the potential to be translated. MKK7 is then specifically phosphorylated in the neurite shaft, where it is part of a MAP kinase signaling module consisting of dual leucine zipper kinase (DLK), MKK7, and JNK1. This triggers Map1b phosphorylation to regulate microtubule bundling leading to neurite elongation. We propose a model in which MKK7 mRNA localization and translation in the growth cone allows for a mechanism to position JNK signaling in the neurite shaft and to specifically link it to regulation of microtubule bundling. At the same time, this uncouples activated JNK from its functions relevant to nuclear translocation and transcriptional activation.

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria.

RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the beta-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the beta-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space.

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

The discovery of SycO highlights a new function for type III secretion effector chaperones.

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.