The RNA-binding protein MARTA2 regulates dendritic targeting of MAP2 mRNAs in rat neurons.

Dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2) in neurons involves a cis-acting dendritic targeting element. Two rat brain proteins, MAP2-RNA trans-acting protein (MARTA)1 and MARTA2, bind to the cis-element with both high affinity and specificity. In this study, affinity-purified MARTA2 was identified as orthologue of human far-upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant-negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule-based motor KIF5 and inhibition of KIF5, but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans-acting factor involved in KIF5-mediated dendritic targeting of MAP2 mRNAs.

Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities.

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1-3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3'-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Shank1 mRNA: dendritic transport by kinesin and translational control by the 5'untranslated region.

Dendritic mRNA transport coupled with local regulation of translation enables neurons to selectively alter the protein composition of individual postsynaptic sites. We have analyzed dendritic localization of shank1 mRNAs; shank proteins (shank1-3) are scaffolding molecules of the postsynaptic density (PSD) of excitatory synapses, which are crucial for PSD assembly and the formation of dendritic spines. Live cell imaging demonstrates saltatory movements of shank1 mRNA containing granules along microtubules in both anterograde and retrograde directions. A population of brain messenger ribonucleoprotein particles (mRNPs) containing shank1 mRNAs associates with the cargo-binding domain of the motor protein KIF5C. Through expression of dominant negative proteins, we show that dendritic targeting of shank1 mRNA granules involves KIF5C and the KIF5-associated RNA-binding protein staufen1. While transport of shank1 mRNAs follows principles previously outlined for other dendritic transcripts, shank1 mRNAs are distinguished by their translational regulation. Translation is strongly inhibited by a GC-rich 5(')untranslated region; in addition, internal ribosomal entry sites previously detected in other dendritic transcripts are absent in the shank1 mRNA. A concept emerges from our data in which dendritic transport of different mRNAs occurs collectively via a staufen1- and KIF5-dependent pathway, whereas their local translation is controlled individually by unique cis-acting elements.

C-terminal prenylation of the CLN3 membrane glycoprotein is required for efficient endosomal sorting to lysosomes.

Mutations in the polytopic lysosomal membrane glycoprotein CLN3 result in a severe neurodegenerative disorder. Previous studies identified two cytosolic signal structures contributing to lysosomal targeting. We now examined the role of glycosylation and the C-terminal CAAX motif in lysosomal transport of CLN3 in non-neuronal and neuronal cells. Mutational analysis revealed that in COS7 cells, CLN3 is glycosylated at asparagine residues 71 and 85. Both partially and non-glycosylated CLN3 were transported correctly to lysosomes. Mevalonate incorporation and farnesyltransferase inhibitor studies indicate that CLN3 is prenylated most likely at cysteine 435. Substitution of cysteine 435 reduced the steady-state level of CLN3 in lysosomes most likely because of impaired sorting in early endosomal structures, particularly in neuronal cells. Additionally, the cell surface expression of CLN3 was increased in the presence of farnesyltransferase inhibitors. Alteration of the spacing between the transmembrane domain and the CAAX motif or the substitution of the entire C-terminal domain of CLN3 with cytoplasmic tails of mannose 6-phosphate receptors have demonstrated the importance of the C-terminal domain of proper length and composition for exit of the endoplasmic reticulum. The data suggest that co-operative signal structures in different cytoplasmic domains of CLN3 are required for efficient sorting and for transport to the lysosome.