Diaz Arturo, Hu Chunbo, Kastner Daniel L, Schaner Philip, Reginato Anthony M, Richards Neil, Gumucio Deborah L
Department of Cell and Developmental Biology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, MI 48109, USA.
Arthritis Rheum. 2004 Nov;50(11):3679-89. doi: 10.1002/art.20600.
To investigate the expression of the familial Mediterranean fever (FMF) gene (MEFV) in human synovial fibroblasts.
MEFV messenger RNA in synovial fibroblasts, chondrocytes, and peripheral blood leukocytes (PBLs) was analyzed by semiquantitative and real-time polymerase chain reaction and ribonuclease protection assay. The subcellular localization of pyrin, the MEFV product, was determined in transfected synovial fibroblasts and HeLa cells with plasmids encoding pyrin isoforms. Native pyrin was detected with an antipyrin antibody.
MEFV was expressed in synovial fibroblasts, but not in chondrocytes. Four alternatively spliced transcripts were identified: an extension of exon 8 (exon 8ext) resulting in a frameshift that predicts a truncated protein lacking exons 9 and 10, the addition of an exon (exon 4a) predicting a truncated protein at exon 5, the in-frame substitution of exon 2a for exon 2, and the previously described removal of exon 2 (exon 2Delta). Exon 8ext transcripts represented 27% of the total message population in synovial fibroblasts. All other alternatively spliced transcripts were rare. Consensus and alternatively spliced transcripts were induced by lipopolysaccharide in synovial fibroblasts and PBLs. In transfected cells, the proteins encoded by all highly expressed splice forms were cytoplasmic. In contrast, native pyrin was predominantly nuclear in synovial fibroblasts, neutrophils, and dendritic cells, but was cytoplasmic in monocytes.
Several MEFV transcripts are expressed and inducible in synovial fibroblasts. A prominent isoform lacks the C-terminal domain that contains the majority of mutations found in patients with FMF. While recombinant forms of all major pyrin isoforms are cytoplasmic, native pyrin is nuclear in several cell types. Thus, mechanisms in addition to splicing patterns must control pyrin's subcellular distribution.
研究家族性地中海热（FMF）基因（MEFV）在人滑膜成纤维细胞中的表达。
采用半定量和实时聚合酶链反应以及核糖核酸酶保护试验分析滑膜成纤维细胞、软骨细胞和外周血白细胞（PBL）中的MEFV信使核糖核酸。用编码吡啉异构体的质粒在转染的滑膜成纤维细胞和HeLa细胞中确定MEFV产物吡啉的亚细胞定位。用抗吡啉抗体检测天然吡啉。
MEFV在滑膜成纤维细胞中表达，但在软骨细胞中不表达。鉴定出四种可变剪接转录本：外显子8延伸（外显子8ext）导致移码，预测产生一种缺少外显子9和10的截短蛋白；添加一个外显子（外显子4a），预测在第5外显子处产生截短蛋白；外显子2a框内替代外显子2；以及先前描述的外显子2缺失（外显子2Delta）。外显子8ext转录本占滑膜成纤维细胞中总信使群体的27%。所有其他可变剪接转录本都很罕见。滑膜成纤维细胞和PBL中脂多糖可诱导共有和可变剪接转录本。在转染细胞中，所有高表达剪接形式编码的蛋白均位于细胞质中。相比之下，天然吡啉在滑膜成纤维细胞、中性粒细胞和树突状细胞中主要位于细胞核中，但在单核细胞中位于细胞质中。
滑膜成纤维细胞中表达并可诱导几种MEFV转录本。一种突出的异构体缺少包含FMF患者中发现的大多数突变的C末端结构域。虽然所有主要吡啉异构体的重组形式都位于细胞质中，但天然吡啉在几种细胞类型中位于细胞核中。因此，除了剪接模式外，还必须有其他机制来控制吡啉的亚细胞分布。
