Transcript mutations of the a regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes
Transcript mutations of the a regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes
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DOI：
10.1016/s0140-6736(02)09966-x
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发表时间：
2002-09-14
期刊：
影响因子：
168.9
通讯作者：
Kammer, GM
中科院分区：
文献类型：
--
作者：
Laxminarayana, D;Khan, IU;Kammer, GM
研究背景系统性红斑狼疮（SLE）是一种自身免疫性疾病，其特征是免疫效应细胞的多种功能障碍，包括增殖和细胞毒性。在SLE患者的T细胞中，由于α和β调节亚基（RI α和RIP）的表达降低，1型蛋白激酶A同工酶的活性大大降低。我们的目的是确定这种同工酶缺乏症的分子机制或机制，通过评估发生突变的RIalpha亚基在SLE.Methods患者的转录本中，我们克隆和测序的RIalpha和相应的基因组DNA的编码区，以检测序列的变化，从8例SLE患者和6名健康对照。由于转录编辑调节腺苷脱氨酶，作用于RNA（阿达尔），我们定量表达的ADAR 1转录在SLE和对照T细胞通过竞争性PCR.Findings的cDNA序列分析显示异质性转录突变，包括缺失，转换和颠换。我们在SLE T细胞中发现了1.22 × 10（-3）/bp的转录本突变，其频率比对照T细胞高7.5倍。相比之下，我们没有发现基因组突变。在来自SLE T细胞的RI α亚基转录物中鉴定出两个热点，一个位于RI α亚基的假底物位点附近，另一个是cAMP结合A结构域的组分。SLE细胞中的ADAR 1 mRNA含量是对照T细胞中的3.5倍（p=0.001）。解释RNA编辑酶可以在RNA的双链区域内将腺苷转化为肌苷，导致转录物突变。该过程可能是导致1型蛋白激酶A的RI α亚基突变的一种机制。
Background Systemic lupus erythematosus (SLE) is an autoimmune disorder characterised by diverse dysfunctions of immune effector cells, including proliferation and cytotoxicity. In T cells from patients with SLE, activity of type 1 protein kinase A isozymes is greatly reduced because of decreased expression of the alpha and beta regulatory subunits (RIalpha and RIP). We aimed to identify a molecular mechanism or mechanisms for this isozyme deficiency by assessing occurrence of mutations in transcripts of the RIalpha Subunit in patients with SLE.Methods We cloned and sequenced cIDNA of RIalpha and corresponding genomic DNA of the coding region to detect sequence changes from eight patients with SLE and six healthy controls. Because transcript editing is regulated by adenosine deaminases that act on RNA (ADAR), we quantified expression of ADAR1 transcripts in SLE and control T cells by competitive PCR.Findings Sequence analyses of cDNA showed heterogeneous transcript mutations, including deletions, transitions, and transversions. We identified 1.22X10(-3)/bp transcript mutations in SLE T cells-a frequency 7.5 times higher than that in control T cells. By contrast, we identified no genomic mutations. Two hotspots were identified in the RIalpha subunit transcripts from SLE T cells, one located adjacent to a pseudosubstrate site of the RIalpha subunit and the other a component of the cAMP binding A domain. ADAR1 mRNA content was 3.5 times higher in SLE cells than in control T cells (p=0.001).Interpretation An RNA-editing enzyme could be converting adenosine to inosine within double-stranded regions of RNA, resulting in transcript mutations. This process could be one mechanism resulting in mutations in the RIalpha subunit of type 1 protein kinase A.