Yasuda Shigemitsu, Wada Seiki, Arao Yukitomo, Kogawa Masakazu, Kayama Fujio, Katayama Shigehiro
Fourth Department of Internal Medicine, Saitama Medical School, Japan.
Endocrinology. 2004 Apr;145(4):1730-8. doi: 10.1210/en.2003-0862. Epub 2004 Jan 8.
Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3' untranslated region (3'UTR). When the 3'UTR of mouse CTR mRNA was labeled by [alpha-(32)P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-binding factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3'UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3'UTR. The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3'UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3'UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3'UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3'UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3'UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3'UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.
我们之前在小鼠破骨细胞中的研究结果表明，降钙素（CT）会改变降钙素受体（CTR）mRNA的稳定性。CTR mRNA转录本在3'非翻译区（3'UTR）含有几个富含腺苷酸/尿苷酸（AU）的不稳定元件。当用[α-(32)P]-尿苷5-三磷酸标记小鼠CTR mRNA的3'UTR时，观察到该转录本与几种细胞提取物之间存在相互作用，包括来自破骨细胞祖代单核细胞/巨噬细胞系RAW 264.7的提取物。相互作用蛋白的分子量范围约为35至50 kDa，类似于富含AU的RNA结合因子1（AUF1）和Hu抗原R（HuR）。放射性标记的3'UTR转录本与一种40 kDa的蛋白结合，该蛋白可从用AUF1 p40转染的细胞中提取。为了证实结合特异性，将含有带有血凝素标签的AUF1 p40的pSG5载体构建体瞬时转染到NIH3T3细胞中。提取物与添加了poly(A)的CTR 3'UTR一起孵育。反应混合物用抗血凝素抗体进行免疫沉淀，沉淀的mRNA种类被提取并用寡聚dT引物进行逆转录。发现针对CTR mRNA序列3'UTR的特异性PCR引物产生了PCR信号。当转染突变的AUF1 p40时未观察到信号。以类似于AUF1结合的方式，还发现HuR与3'UTR结合。在从小鼠破骨细胞提取的RNA中也发现了AUF1 p40和HuR的特异性结合。用CT处理破骨细胞不会显著影响AUF1的表达，但会降低HuR及其mRNA的水平。通过在四环素调控系统的控制下表达含有或不含有CTR 3'UTR的荧光素酶报告构建体，进一步测试了CTR 3'UTR在mRNA稳定性中的作用。结果表明，添加3'UTR显著缩短了荧光素酶报告基因的mRNA半衰期。这些结果表明，AUF1 p40、HuR和CTR mRNA转录本的3'UTR可能参与CTR mRNA表达的转录后调控。
