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Mechanisms of kinesin-dependent RNA transport and translational regulation

Periodic Reporting for period 1 - RNA Transport and Control (Mechanisms of kinesin-dependent RNA transport and translational regulation)

Periodo di rendicontazione: 2015-06-01 al 2017-05-31

Context and state of the art
mRNA transport in general: Cytoplasmic mRNA transport and local translation are essential for many processes requiring symmetry breaking such as embryonic development, cell migration and neuronal differentiation (1). They allow cells to configure protein networks locally and exclude proteins from locations where their activity is harmful. mRNAs are transported along microtubules (MTs) by different kinesins and cytoplasmic dynein and get anchored at the actin cortex, intermediate filaments or unknown structures (1, 3). Extrinsic cues or unknown events activate translation when required (4). mRNA localisation requires primary sequences and secondary structures often localised in their 3’UTR (3). RNA binding proteins recognize these sequences and assemble with their mRNA target into mRNPs. It is thought that motor or adaptor proteins recognise different features on the surface of mRNPs with different affinities. This defines the extent to which transport of an mRNP is biased towards one direction, which ultimately gives rise to the steady state distributions pattern of mRNAs observed (5). Since the first discovery of cytoplasmic mRNA transport almost three decades ago, several essential questions could not be answered mostly due to the complexity of the in vivo situation and the approaches available. It is still not known which RBPs or adaptor proteins are essential to recruit microtubule-binding motor proteins to mRNPs and to which extend this varies between different transported mRNAs. In more general terms, the whole nature of the mRNP-motor interface remains a mystery.

mRNA transport in neurons: In neurons, thousands of mRNAs are transported into axons or dendrites by so far not identified transport mechanisms (6). Localised translation allows neurons to react to incoming stimuli instantly by locally producing proteins, which is a requirement for long-term memory formation and maintenance. Mutations affecting zipcodes, RBPs or motor-proteins required for neuronal mRNA localization were shown to lead to severe neurodegenerative diseases as ALS, FXTAS and FXS (7), underlining the need to understand the mechanisms that drive neuronal mRNA transport. Neuronal mRNA transport occurs in packages of single to a few copies of mRNAs (8-11), mostly in a translationally repressed mode. A good example is the CaMKIIa-mRNA, which encodes the a-subunit of the Ca2+-calmodulin kinase II. Dendritic localisation of CaMKIIa-mRNA requires its 3’UTR harbouring binding sites for RBPs as FMRP and Staufen2, which are required for its correct localisation. After induction of long-term potentiation, CaMKIIa-mRNA is transported to distal parts of dendrites where its translation is locally regulated (12, 13). Inhibiting the localisation of this mRNA leads to a significant reduction of CamKIIa protein at postsynaptic densities and a strong reduction of cognitive abilities in animal models (12). Also in this specific case, it is not understood how RBPs, potential adaptors and motor proteins, which are essential for the transport of CaMKIIa-mRNA are mechanistically contributing to its correct localisation.

The central goal of this project is the ‘In vitro reconstitution of kinesin-dependent RNA transport’. While it is clear, that RNA distribution patterns in neurons are created by active transport processes of mRNPs along microtubules, the essential enzymatic activities required are not known. Before we can understand how overall distributions of thousands of mRNA are generated, we first need to understand how any specific mRNA can be transported along microtubules – a question unanswered since the first MT based mRNA transport was observed more than 2 decades ago.
Accomplishments
In order to achieve the central goal we focussed on a set of proteins: Kif3A, Kap3, APC and a specific mRNA: Tubb2b mRNA. It was reported that APC can be transported by the Kif3A-Kap3 complex in mouse neurons (14). In 2014 it was shown that APC binds mRNAs localised to axonal growth cones with Tubb2b-mRNA being one of its targets (15). Hence, we speculated that APC could be the adaptor needed to link some mRNAs to a kinesin-based transport complex. To test this hypothesis, I purified all needed proteins and in vitro transcribed the Tubb2b-mRNA 3’UTR, which harbours a G-rich APC-binding motif. We tested the functionality of purified APC-GFP by measuring its binding to Kap3 and Tubb2b-RNA by MST (Microscale Thermophoresis). APC-GFP binds to an in vitro transcribed Tubb2b-3’UTR-mRNA fragment with low nanomolar affinity which is close to the published value found by gel-shift assays (15). Kap3 binds APC-GFP with an affinity in the high nanomolar range. As it is not evident from existing literature whether Kif3A can homo-dimerise or hetero-trimerise with Kap3, we performed size-exclusion-coupled multi-angle-light scattering (SEC-MALS) experiments. Kap3 forms a monomer of 80kDA size while Kif3A dimerises to form a 160kDa complex. When combined, a new peak with a mass of 240kDa appears, which shows that the Kif3A-Kap3 complex assembles at a stoichiometric ratio of 2:1. We next tested whether the purified Kif3A-Kap3-GFP complex was active by using TIRF-M coupled in vitro motility assays on Taxol-stabilized, immobilised microtubules (MTs). Processive movement of Kif3A-Kap3-GFP complexes could be observed. Subsequently, we tested whether the APC-Kap3 interaction are strong enough to allow processive transport of APC-GFP in complex with the Tubb2b-RNA fragment. APC-GFP can complex its target mRNA and bind to Kif3A-Kap3 at the same time, allowing processive mRNA transport. The described experiments show the very first reconstitution of a microtubule based mRNA transport system (Fig. 1) and we anticipate that this pioneering work will open a new field allowing to dissect the core mechanisms driving cytoplasmic mRNA distribution.

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With the in vitro reconstitution of neuronal kinesin-based RNA transport a new field is opened which enables a molecular and mechanistic investigation of molecular processes underlying synaptic plasticity which is the basis for learning and memory formation. This project will lead to a better understanding of neuronal RNA transport and hopefully to a better understanding of mental diseases like Fragile X Syndrome or Autism Spectrum disorders that have been linked to dysfunction in neuronal RNA transport.