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

Ca²⁺ imaging of photoreceptor terminals.

**<p>p<0.01;</p>*<p>p<0.05; n.s., not significant. Significance levels were determined by Kruskal-Wallis ANOVA.</p

Photoreceptor degeneration in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Quantification of the percentage of TUNEL positive cells in the ONL from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). <b>B:</b> Immunocytochemical staining of GFAP on P28 old wild-type, ΔEx14–17, and I756T mutant retina shows more pronounced Müller cell reactivity in the I756T mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm.</p

Age-dependent ONL thickness in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Labeling of nuclei with DAPI (blue) and of cone photoreceptor outer segments and terminals (arrowheads) with peanut agglutinin (green) on retinal cryostat sections of wild-type, ΔEx14–17, and I756T mutant mice at 8 months. <b>B:</b> Quantification of the number of cell rows in the outer nuclear layer (ONL) from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values in are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). POS, photoreceptor outer segments; OPL, outer plexiform layer. Scale bar: 10 µm.</p

Comparison of the sprouting phenotype in the wild-type, ΔEx14–17, and I756T mutant retinae.

<p><b>A–C:</b> Immunocytochemical triple staining of Calbindin (red), PKCα (green), and VGluT1 (blue) on P28 old wild-type (A), ΔEx14–17 (B), I756T (C) outer retinae shows sprouting of ON-bipolar cell dendrites as well as horizontal cell processes into the ONL of both <i>Cacna1f</i> mutants. The VGluT1 labeling shows the existence of presynaptic contacts with the sprouting elements. <b>D:</b> Comparison of the severity of horizontal cell sprouting in the wild-type, ΔEx14–17, I756T mutant outer retina at P6, P14, P28, two months, and eight months. Asterisks indicate the onset of noticeable sprouting in the <i>Cacna1f</i> mutants. In the I756T mutant retina, noticeable horizontal cell sprouting started earlier (P14) than in the ΔEx14–17 mutant retina (P28), but declined at eight months, when sprouting still continued in the ΔEx14–17 mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar in A for A–C<b>:</b> 10 µm; in D: 20 µm.</p