Identification and characterization of seven new exon 11-associated splice variants of the rat μ opioid receptor gene, OPRM1.
Identification and characterization of seven new exon 11-associated splice variants of the rat μ opioid receptor gene, OPRM1.
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DOI：
10.1186/1744-8069-7-9
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发表时间：
2011-01-21
期刊：
影响因子：
3.3
通讯作者：
Pan YX
中科院分区：
文献类型：
--
作者：
Xu J;Xu M;Rossi GC;Pasternak GW;Pan YX
小鼠mu阿片受体（OPRM1）基因在基因的3‘端和5’端经历了广泛的选择性剪接。此前，在大鼠OPRM1基因中已经发现了几个通过3'剪接产生的c端变异。在小鼠和人类中，5'剪接产生了许多包含外显子11的变体。外显子11敲除小鼠的研究表明，这些外显子11相关变异在介导一类mu阿片样物质的镇痛作用方面具有重要的功能，包括吗啡-6β-葡糖苷（M6G）和海洛因，但不包括吗啡和美沙酮等其他阿片样物质。我们现在已经研究了大鼠的5'剪接。目前的研究在大鼠中发现了一个同源外显子11和7个外显子11相关变异，表明外显子11及其相关变异在小鼠、大鼠和人类中都是保守的。RT-PCR显示，这些变体在几个大脑区域的表达存在显著差异，这意味着外显子11相关变体的mRNA加工具有区域特异性。在7个大鼠外显子11相关变异中，4个编码与rmor1中发现的相同的蛋白质，2个预测6 TM变异，1个rmor1h2产生了一个新的n端变异，其中在先前建立的rmor1序列的n端存在额外的50个氨基酸。当在CHO细胞中表达时，rmoor - 1h2中额外的50个氨基酸的存在显著改变了激动剂诱导的G蛋白激活，而对阿片结合的影响很小。大鼠外显子11及其相关变异的鉴定进一步证明了OPRM1基因在啮齿类动物和人类中5'剪接的保守性。这些外显子11相关变异的功能相关性是通过其mrna的区域特异性表达和n端序列对激动剂诱导的G蛋白偶联的影响来提示的。外显子11敲除小鼠中外显子11相关变异在M6G和海洛因镇痛小鼠中的重要性表明，这些类似的大鼠变异也可能在大鼠中发挥类似的作用。大鼠OPRM1基因的选择性剪接所产生的复杂性可能为理解大鼠对各种mu阿片样物质的不同反应提供重要见解。
The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat. The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding. The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various mu opioids seen in rats.
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