13 research outputs found
BC1-FMRP interaction is modulated by 2ā²-O-methylation: RNA-binding activity of the tudor domain and translational regulation at synapses
The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control. One component of BC1-containing RNP complexes is the fragile X mental retardation protein (FMRP) that is implicated in translational repression. Peptide mapping and computational simulations show that the tudor domain of FMRP makes specific contacts to BC1 RNA. Endogenous BC1 RNA is 2ā²-O-methylated in nucleotides that contact the FMRP interface, and methylation can affect this interaction. In the cell body BC1 2ā²-O-methylations are present in both the nucleus and the cytoplasm, but they are virtually absent at synapses where the FMRPāBC1āmRNA complex exerts its function. These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets. We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP. All together these findings provide further insights into the translational regulation by the FMRPāBC1 complex at synapses
Efficient Transamination under Mild Conditions:Ā Preparation of Primary Amine Derivatives from Carbonyl Compounds via
Specificity and Redundancy of Profilin 1 and 2 Function in Brain Development and Neuronal Structure
Profilin functions have been discussed in numerous cellular processes, including actin polymerization. One puzzling aspect is the concomitant expression of more than one profilin isoform in most tissues. In neuronal precursors and in neurons, profilin 1 and profilin 2 are co-expressed, but their specific and redundant functions in brain morphogenesis are still unclear. Using a conditional knockout mouse model to inactivate both profilins in the developing CNS, we found that threshold levels of profilin are necessary for the maintenance of the neuronal stem-cell compartment and the generation of the differentiated neurons, irrespective of the specific isoform. During embryonic development, profilin 1 is more abundant than profilin 2; consequently, modulation of profilin 1 levels resulted in a more severe phenotype than depletion of profilin 2. Interestingly, the relevance of the isoforms was reversed in the postnatal brain. Morphology of mature neurons showed a stronger dependence on profilin 2, since this is the predominant isoform in neurons. Our data highlight redundant functions of profilins in neuronal precursor expansion and differentiation, as well as in the maintenance of pyramidal neuron dendritic arborization. The specific profilin isoform is less relevant; however, a threshold profilin level is essential. We propose that the common activity of profilin 1 and profilin 2 in actin dynamics is responsible for the observed compensatory effects
La protein has a positive effect on the translation of TOP mRNAs in vivo
In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5ā²-untranslated region (5ā²-UTR), including a stretch of pyrimidines at the 5ā²-end. The 5ā²-terminal oligopyrimidine (5ā²-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5ā²-UTR downstream from the 5ā²-TOP sequence
KIF1BĪ² transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner
KIF1BĪ² is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1BĪ² isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1BĪ² (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1BĪ² localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1BĪ² is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1BĪ² forms RNA-protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as PurĪ±, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1BĪ² and Calmodulin is Ca(+2)-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1BĪ² at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1BĪ² is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1BĪ², its interaction with Ca(2+) sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways
KIF1B beta transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner
KIF1BĪ² is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1BĪ² isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1BĪ² (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1BĪ² localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1BĪ² is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1BĪ² forms RNA-protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as PurĪ±, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1BĪ² and Calmodulin is Ca(+2)-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1BĪ² at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1BĪ² is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1BĪ², its interaction with Ca(2+) sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.status: publishe
The actin-binding protein profilin 2 is a novel regulator of iron homeostasis
Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 39 untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice.This work was supported by grant SAF2015-70412-R from the Spanish Secretary of Research, Development, and Innovation (Ministerio de EconomĆa, Industria y Competitividad [MINECO]) and grant DJCLS R14/04 from Deutsche JosĆ© Carreras LeukƤmie Stiftung, grant 2014 SGR225 (Grups de Recerca Emergent, from Generalitat de Catalunya) from Generalitat de Catalunya, and financial support from FundaciĆ³ Internacional Josep Carreras and from Obra Social āla Caixaā Spain (M. Sanchez). All work on the Pfn2 KO mouse model was supported by the Deutsche Forschungsgemeinschaft grant SFB1089 and SPP1464 (W.W.). S.L. was supported by the EMBO Short Term Fellowship ASTF 301-2013 for her work on the Pfn2ā/ā mice at the Institute of Genetics, University of Bonn, Bonn, Germany
The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP
Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.status: publishe