Tra2B Regulates The Expression Of RGS4, A Signal Mediator Involved In Morphine Addiction

TRA2B is a typical member of SR (Serine/Arginine-rich proteins)-related proteins which can regulate gene expression via affecting alternative splicing. It has been reported that TRA2B is ubiquitously expressed in the brain and participates in many physiological and pathological processes. In the present study, for further understanding of the functions of TRA2B in the brain, we utilized multiple experimental methods including Western blot, IHC and immunofluoresence to study the regional-specific expression pattern of TRA2B in adult rat brain. Two status of post-translational modification of TRA2B, including phosphorylation and non-phosphorylation-related modification were detected, and the phosphorylation of TRA2B is coordinated to its nuclear localization. Although expressed ubiquitously, the total TRA2B protein levels and the ratio of phosphorylated and non-phosphorylated TRA2B proteins varied in different brain regions, suggesting a regional-specific manner. Besides, TRA2B proteins also showed a cell type specific distribution pattern:detected only in NeuN positive neurons, but not in GFAP positive astrocytes. Confocal microscopy revealed a speckled staining pattern of TRA2B in the nuclei of neurons. In addition, we found for the first time that TRA2B positive cells were also present in olfactory bulb, inferior colliculus, superior colliculus, midbrain, cerebellum Purkinje cell and granule cell layer.Noticeably, by detailed analysis of the IHC results, we found that intensive staining was present in several brain regions closely related to drug abuse, such as locus coeruleus (LC), ventral tegmental area (VTA), nucleus accmbens (NAc), prefrontal cortex (PFC), reticulotegmental pontine nucleus (RtTg) and dorsal central gray (CGD), suggesting that TRA2B may be involved in drug abuse. According to the fact that TRA2B is a regulator of gene expression, we predict that TRA2B is possibly involved in regulating the expression of some drug abuse-related genes in these brain nuclei.To test this possibility, we assumed that if TRA2B is involved in regulating the expression of drug abuse-related genes, it will have a similar distribution pattern to these gene products. To verify this assumption, we studied the co-distribution of TRA2B and those drug abuse-related signal mediators by immunofluorecence and confocal microscopy in the drug-related nuclei. We found that TRA2B was co-localized with RGS4, an important signal mediator in drug addiction, in many drug-related nuclei, such as PFC, NAc, LC and CGD. The close distribution correlation between TRA2B and RGS4in the brain suggests a potential functional correlation between them.As a typical SR protein, TRA2B largely functions as a binding protein to mRNA and also to other splicing factors, thereby contributing to spliceosome assembly and splicing site recognition. Accordingly, to study the functional relation between TRA2B and RGS4, we carried out immuno-precipitation assay to find TRA2B-associated mRNA and proteins. As we expected, endogenous RGS4mRNA indeed exists in TRA2B immuno-precipitated complex. Two other proteins were also found in TRA2B pulldown products. Mass Spectrometry analysis showed that they were ASF/SF2and SRp30c, two other members of the SR protein family known to interact with TRA2B. The results suggest the likelihood that together with other SR proteins, TRA2B exists in RGS4pre-mRNA spliceosome and regulates its splicing.To test whether RGS4gene expression is regulated by TRA2B, we examined the changes of RGS4mRNA and protein levels in cells transfected with TRA2B over-expression plasmids or TRA2B specific siRNAs, by RT-PCR and Western blot analysis. The results showed that RGS4mRNA and proteins were significantly reduced by TRA2B silence, and increased by TRA2B over-expression in a concentration dependent manner, suggesting that RGS4expression is regulated by TRA2B.Then we pursued to determine the critical structures required for TRA2B to regulate RGS4splicing. It has been reported that the splicing function of SR proteins is related to its sub-nuclear distribution, which depends mainly on its RS domains. So we took advantage of GFP reporter gene to observe the critical motifs of TRA2B for its sub-nuclear distribution. The results showed that the nuclear localization of TRA2B depends on its RS1domain, but not the RS2domain, suggesting the functional difference between the two RS domains. There were several nuclear speckles localization signals on RS1domain, locating on56-68aa,79-89aa,89-99aa regions, suggesting that these redundant signals provide a tight control of TRA2B distribution in nuclear speckles.Nowadays, many studies have demonstrated that drug stimulation affect the expression levels of RGS4in drug-related brain nuclei. Considering the results mentioned above that TRA2B is highly expressed in drug-related nuclei and co-localized with RGS4, and that TRA2B binds to RGS4mRNA and regulates RGS4expression, we proposed that TRA2B is likely involved in the changes of RGS4caused by drug stimulation in a pattern similar to that observed in the cultured cells as descrbed above. To examine this possibility, we then investigated the effect of drug stimuli on TRA2B. The results showed that compared with saline injection, TRA2B and RGS4protein levels were decreased after acute morphine injection and increased after chronic morphine administration. Meanwhile, administration of naloxone, the opioid receptor antagonist, prevented these changes. These results are consistent with those from the TRA2B over-expressed or silenced cells, suggesting that TRA2B is involved in regulation of RGS4in drug stimulus.In conclusion, the present studies demonstrated that TRA2B regulates the expression of RGS4, a signal mediator involved in morphine addiction. In the studies, we investigated the co-localization of TRA2B and RGS4in drug addiction-related brain nuclei, and the correlated changes of their expression under acute and chronic morphine stimuli. Moreover, protein structural basis for TRA2B to regulate RGS4expression was examined. The results suggest that as a splicing regulator with multiple functions in the brain, TRA2B is likely involved in drug addiction signal pathway via regulating the expression of a signal mediator RGS4.