Pulcrano G, Leonardo R, Piscopo M, Nargi E, Locascio A, Aniello F, Branno M, Fucci L
Department of Structural and Functional Biology, University of Naples Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia, Naples, Italy.
Gene. 2007 Dec 30;406(1-2):124-33. doi: 10.1016/j.gene.2007.07.018. Epub 2007 Jul 31.
In P. lividus sea urchin the H3.3 histone variant is coded by an mRNA characterized by a long 3'UTR containing ARE (AU-Rich element) motifs. RNA stability assays performed in rabbit reticulocyte lysate showed that such 3'UTR affects the degradation rate of the transcripts. In fact, chimeric molecules containing the 3'UTR of H3.3 transcript, ligated to the coding region of the rabbit beta-globin transcript, were unstable whereas chimeric molecules containing mainly the coding region of the H3.3 transcript were stable as the wild-type globin mRNA. Three proteins (45kDa, 32kDa and 25kDa) that bind specifically the 3'UTR have been revealed in the whole protein extracts of embryos at different stages of development. PLAUF, a P. lividus RNA-binding protein similar to human and rodent AUF1 proteins, was identified as the 32kDa factor using anti-PLAUF antibody in Western blot and supershift mobility assays. Moreover the recombinant GST-PLAUF protein specifically binds part of the H3.3 3'UTR and in vitro affects the half-life of the transcript. In addition in situ hybridization experiments demonstrated that PLAUF and H3.3 histone mRNAs co-localize in embryos at different stages of development. In conclusion all the reported results suggest that PLAUF can bind in vivo the 3'UTR of the H3.3 histone mRNA and plays some role in the stability of the mRNA.
在紫球海胆中，H3.3组蛋白变体由一种mRNA编码，该mRNA的特征是具有一个长的3'UTR，其中包含富含AU元件（ARE）基序。在兔网织红细胞裂解物中进行的RNA稳定性分析表明，这种3'UTR会影响转录本的降解速率。事实上，含有H3.3转录本3'UTR并连接到兔β-珠蛋白转录本编码区的嵌合分子不稳定，而主要含有H3.3转录本编码区的嵌合分子则与野生型珠蛋白mRNA一样稳定。在不同发育阶段胚胎的全蛋白提取物中发现了三种能特异性结合3'UTR的蛋白质（45kDa、32kDa和25kDa）。使用抗PLAUF抗体通过蛋白质免疫印迹和超迁移率分析，将一种与人和啮齿动物AUF1蛋白相似的紫球海胆RNA结合蛋白PLAUF鉴定为32kDa因子。此外，重组GST-PLAUF蛋白能特异性结合H3.3 3'UTR的一部分，并在体外影响转录本的半衰期。另外，原位杂交实验表明，PLAUF和H3.3组蛋白mRNA在不同发育阶段的胚胎中共定位。总之，所有报道的结果表明，PLAUF在体内能结合H3.3组蛋白mRNA的3'UTR，并在mRNA的稳定性中发挥一定作用。
