12 research outputs found
Isolation and characterization of alternatively spliced variants of the mouse sigma<sub>1</sub> receptor gene, Sigmar1
<div><p>The sigma<sub>1</sub> receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma<sub>1</sub> receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma<sub>1</sub> receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma<sub>1</sub> receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma<sub>1</sub> receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.</p></div
Heterodimerization of mMOR-1 and mSigmar1 splice variants.
<p>Flag-tagged mMOR-1 and HA-tagged mSigmar1 variants were transiently co-transfected into HEK293 cells. Co-IP was performed as described in Materials and Methods. Briefly, cleared whole cell lysate was incubated with EZview Red Anti-Flag or anti-HA Affinity gels overnight. After washing, the tagged proteins on the affinity gels were eluted with 3xFlag or HA peptide, and the elutes were analyzed in Western blot. Top panel: Immunoblot (IB) with anti-HA antibody using elutes from immunoprecipitation (IP) with EZview Red Anti-Flag Affinity Gel. Bottom panel: IB with anti-Flag antibody using elutes from IP with EZview Red Anti-HA Affinity Gel. The representative blots from two independent experiments were shown.</p
Alignment of the predicted amino acid sequences of the mouse Sigmar1 splice variants.
<p>The predicted amino acid sequences were aligned using ALIGN progrom in Vector NTI (Invitrogen). The exon assignment for the predicted amino acids is shown by colored letters and backgrounds. The transmembrane domain is underlined. The number of amino acids is list on the right. *: stop codon. The nucleotide and deduced amino acid sequences of the mouse sigma1 receptor 1a (mSIG-1A), mSIG-1B, mSIG-1C, mSIG-1D and mSIG-1E have been deposited in the GenBank database with Accession numbers: AY390764, AY390765, AY390766, AY390767 and AY390768, respectively.</p
Effects of mSigmar1 splice variants on physical association of the original mSIG-1 with mMOR-1.
<p>A). Western blot of HA antibody-precipitated elutes with Flag antibody (ab). Equal amounts of mSIG-1/HA (1.2 μg) and mMOR-1/Flag (1.2 μg) were co-transfected into a 100 mm plate of HEK293 cells, together with each untagged mSigmar1 variant at four different amounts (0 μg, 1.2 μg, 2.4 μg or 3.6 μg, labeled as 0, 1, 2, 3, respectively), by using Effectene (Qiagen) as described in Materials and Methods. Appropriate amount of pcDNA3 vector DNA was added to maintain the total amount of DNA in each transfection as 6 μg. Whole cells were solubilized 48 hr after transfection, and cleared lysate by centrifugation was then used in immunoprecipitation with EZview HA affinity gel. Eluted proteins by Flag peptides were separated by SDS-PAGE and transferred into a PVDF membrane, which was then immunobloted with an anti-Flag antibody, as described in Materials and Methods. The representative blot from two independent experiments was shown. IP: immunoprecipitation; IB: immunoblot. B). Western blot of HA-antibody-precipitated fraction with EZview HA affinity gel. The same elutes were analyzed using an anti-HA antibody in immunobloting. C) & D). Western blot of cleared lysate with anti-Flag antibody or anti-HA antibody. Cleared lysate samples before IP were analyzed using anti-Flag antibody (C) or an anti-HA antibody (D) in immunobloting.</p
Expression of individual mouse Sigmar1 splice variant mRNAs.
<p>Each panel represents one of the mouse Sigmar1 splice variants. Bars represent the mean of 2<sup>-ΔC(t)</sup> values ± S.E.M. from at least three independent samples in one experiment. ΔC(t) values were determined as: Δ<i>C</i>(<i>t</i>) = <i>C</i>(<i>t</i>)<i>variant</i> − <i>Normalized factor (NF)</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174694#pone.0174694.ref033" target="_blank">33</a>]. PFC: prefrontal cortex. Significant difference was calculated by One-way ANOVA with Tukey’s multiple comparisons test (Prism 6.0). The results of the statistical analysis were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174694#pone.0174694.s005" target="_blank">S2 Table</a>.</p
Overall expression levels of the mouse Sigmar1 splice variant mRNAs.
<p>Expression levels (-1/ΔC(t)) of the mouse Sigmar1 splice variant mRNAs is plotted across tissues and brain regions using MS excel. ΔC(t) values were determined as: Δ<i>C</i>(<i>t</i>)<i>variant</i> − <i>Normalized factor (NF)</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174694#pone.0174694.ref033" target="_blank">33</a>] from at least three independent samples in one experiment. PFC: prefrontal cortex. SYBR green qPCR was performed as described in Materials and Methods. All the primer sequences and qPCR conditions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174694#pone.0174694.s004" target="_blank">S1 Table</a>.</p
Expression levels of selected ten splice variants in brain regions of four mouse strains.
<p>Expression levels of (−1)/(Log<sub>2</sub> (E<sup>−ΔC(t)</sup>)) of selected variants is plotted across regions using MS excel. Pfc: prefrontal cortex; Str: striatum; Tha: thalamus; Hyp: hypothalamus; Hip: hippocampus; PAG: periaqueductal gray; BS: brainstem; Cb: cerebellum; Spc: spinal cord; WB: whole brain. 129∶129P3/J; B6: C56BL/6J; SJL: SJL/J; SWR: SWR/J.</p
Heatmap of expression levels of <i>OPRM1</i> splice variant mRNAs.
<p>The heatmap was generated by clustering Z-scores calculated from the values (Log<sub>2</sub> (E<sup>−ΔC(t)</sup>) of individual variants across different strains/regions using R statistical language (2.15.0) (<a href="http://www.r-project.org" target="_blank">www.r-project.org</a>), and plotting based upon expression levels among the variants with enhanced heatmap function heatmap.2 from gplots package. Pfc: prefrontal cortex; Str: striatum; Tha: thalamus; Hyp: hypothalamus; Hip: hippocampus; PAG: periaqueductal gray; BS: brainstem; Cb: cerebellum; Spc: spinal cord; WB: whole brain. 129∶129P3/J; B6: C56BL/6J; SJL: SJL/J; SWR: SWR/J. Expression level was indicated by Z-score.</p
Region-specific expressions of ten-selected <i>OPRM1</i> splice variants.
<p>Each panel represents the regional expression of one variant in four inbred mouse strains. Red bar: B6 mice; Green bar: 129 mice; Blue bar: SJL mice; Brown bar: SWR mice. Bars represent the mean of E<sup>−ΔC(t)</sup> values ± S.E.M. Pfc: prefrontal cortex; Str: striatum; Tha: thalamus; Hyp: hypothalamus; Hip: hippocampus; PAG: periaqueductal gray; BS: brainstem; Cb: cerebellum; Spc: spinal cord; WB: whole brain. mE1-2 represents all full-length 7-TM variants. Significant difference was calculated by Two-way ANOVA with Tukey’s multiple comparisons test for each variant (Prism 5.0). The results of the statistical analysis were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s008" target="_blank">Table S3</a>. The expression profiles of the other 19 <i>OPRM1</i> splice variants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s001" target="_blank">Figures S1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s002" target="_blank">S2</a>.</p
Strain-specific expressions of <i>OPRM1</i> splice variants.
<p>Each panel represents the expression of one variant in 10 regions of 4 inbred mouse strains. Red bar: B6 mice; Green bar: 129 mice; Blue bar: SJL mice; Brown bar: SWR mice. Bars represent the mean of E<sup>−ΔC(t)</sup> values ± S.E.M. Pfc: prefrontal cortex; Str: striatum; Tha: thalamus; Hyp: hypothalamus; Hip: hippocampus; PAG: periaqueductal gray; BS: brainstem; Cb: cerebellum; Spc: spinal cord; WB: whole brain. Significant difference was calculated by Two-way ANOVA with Tukey’s multiple comparisons test for each variant. The results of the statistical analysis were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s008" target="_blank">Table S3</a>. The expression profiles of the other 19 <i>OPRM1</i> splice variants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s003" target="_blank">Figures S3</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111267#pone.0111267.s004" target="_blank">S4</a>.</p