Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome

A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1-/- mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities

Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome

<div><p>A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1^-/- mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities. </p> </div

Simiate.

<p>A) Co-immunoprecipitation of FMRP and Simiate- or ARMC1-mRNA visualized by RT-PCR. IgG serves as negative control for the assay. B) The same experiment as shown in A, but the immunoprecipitation of FMRP is implemented with FMR1^-/- mice. C) Negative controls for the reaction showing that the association requires not only FMRP (cp. panel B), but also transcribed Simiate-mRNA. </p

The expression of Simiate.

<p>A) Simiate is present in several different tissues. Endogenous Simiate was immunoprecipitated from different mouse organs using rbαSimiate and subjected to western blotting. Pre-immune serum (Pre-IS) served as negative control. Differences in the amount of protein available to immunoprecipitation (“input”) are displayed as total quantities of proteins normalised to the sample containing the highest quantum of protein (liver). B) Representative immunofluorescence pictures demonstrating the expression of endogenous Simiate during the development of primary hippocampal neurons. The numbers indicate the corresponding day in vitro (div). At the bottom, magnifications of a dendrite (a) and a nucleus (b) are shown. </p

Simiate and nuclear speckles in FMR1^-/- mice.

<p>A,B) The graphs show the volume (Vol.; A,C) and distribution (Dist.; B,D) of nuclear Simiate in neuronal and non-neuronal cells for diverse brain regions from FMR1^-/- and wildtype mice. Neurons were identified by the presence of NeuN. A,B) In each column, symbols indicate the median, while the error bars display the corresponding 10/90 quantile. Stars represent significant differences between medians, clubs between variances. Each group contains 14-18 cells (n) from two independent experiments. The distribution was calculated as ratio of surface to volume. Please note the logarithmic scale in A). Results from Dunn's multiple comparison post-test of Kruskal-Wallis statistics for A) and B) are shown in C) and D), respectively. C, D) Yellow backgrounds indicate significant differences between wildtype and FMR1^-/-. CA1,3: regions of the Hippocampus, Cor: Cortex, CPu: Caudoputamen, ns: non significant, PC: Purkinje cell, wt: wildtype.</p

Simiate in neuronal and non-neuronal nuclei of an adult FMR1^-/- mouse brain.

<p>A) A part of the pyramidal cell layer of the Hippocampus. Neuronal cells are marked with NeuN. The nuclei of two glia cells located at the bottom of the picture are delineated with dotted lines in all graphs not displaying DAPI. B) 3D reconstruction of a neuronal nucleus (red box in A). NeuN is not only a marker for neuronal cells, but also known to reside in nuclear speckles.</p

Simiate is vital to cells.

<p>A) Chariot reagent shuttled rbαSimiate (0.5µg) detects FLAG-Simiate in transfected HEK-293 cells. The nuclei are visualized by DAPI staining. The arrows indicate clusters of rbαSimiate and FLAG-Simiate immunofluorescence inside the nucleus. B-D) Apoptosis in rbαSimiate and αrbAlexa568 treated HEK-293 cells. B) 0.25µg rbαSimiate, C) 1.0µg rbαSimiate and D) 1.0µg αrbAlexa568 as negative control. TUNEL staining (in green) served to identify apoptotic cells, while nuclear speckles were outlined with SC35 (in red). E) Quantification of the amount of endogenous Simiate epitopes not targeted by antibodies. F) Quantification of apoptotic cells. The increase in the percentage of TUNEL positive cells after rbαSimiate treatment is extremely significant (Chi²: p<0.001) compared to the control treatment, where rbαAlexa568 was applied. n (0.25-2.0µg) rbαAlexa568: 293, 262, 276 and 276 cells and n (0.25-2.0µg) rbαSimiate: 300, 237, 241 and 252 cells. The two bottom graphs show the volume (G) and distribution (H) of nuclear speckles in rbαSimiate (filled symbols) and αrbAlexa568 (empty symbols) treated HEK-293 cells. 0.25µg antibodies are shown in light gray, whereas 0.5µg are demonstrated in dark gray. Due to massive apoptosis induced by higher amounts of rbαSimiate, those cells were not analysed. n (0.25-2.0µg) rbαAlexa568: 25, 25, 20 and 20 cells and n (0.25-0.5µg) rbαSimiate: 24 and 25 cells. Stars represent significant differences between medians.</p

Simiate and the focal adhesion kinase FAK1 cooperate in the regulation of dendritogenesis

Despite the crucial importance of dendritogenesis for the correct functioning of neurons, the molecular mechanisms underlying neuronal arborisation are still not well understood. Current models suggest that distinct parts and phases of dendritic development are regulated by the expression of distinct transcription factors, that are able to target the cytoskeleton. Two proteins recently implicated in dendritogenesis are the Focal Adhesion Kinase FAK1 and the Actin-binding protein Simiate. Using heterologous expression systems as well as mouse brain extracts in combination with coprecipitation assays, we show that Simiate is able to associate with FAK1. Differential centrifugation experiments further revealed the interaction to be present in cytosolic as well as nuclear fractions. Inside the nucleus though, Simiate preferentially binds to a FAK1 isoform of 80 kDa, which has previously been shown to regulate transcription factor activity. Investigating the function of both proteins in primary hippocampal cultures, we further found that FAK1 and Simiate have distinct roles in dendritogenesis: While FAK1 increases dendrite length and number, Simiate preferentially enhances growth and branching. However, if being confined to the nucleus, Simiate selectively triggers primary dendrite formation, enhancing transcription activity at the same time. Since the effect on primary dendrites is specifically re-normalized by a co-expression of FAK1 and Simiate in the nucleus, the data implies that the two proteins interact to counterbalance each other in order to control dendrite formation. Looking at the role of the cytosolic interaction of FAK1 and Simiate, we found that neurotrophin induced dendritogenesis causes a striking colocalisation of FAK1 and Simiate in dendritic growth cones, which is not present otherwise, thus suggesting that the cytosolic interaction stimulates growth cone mediated dendritogenesis in response to certain external signals. Taken together, the data show that FAK1 and Simiate exert several and distinct actions during the different phases of dendritogenesis and that these actions are related to their subcellular localisation and their interaction