Pulcrano G, Leonardo R, Aniello F, Mancini P, Piscopo M, Branno M, Fucci L
Department of Genetics, General and Molecular Biology, University of Naples Federico II, Via Mezzocannone 8, 80134, Naples, Italy.
Gene. 2005 Feb 28;347(1):99-107. doi: 10.1016/j.gene.2004.12.006.
Preliminary results have shown that various proteins bind long 3'UTR of the transcript for Paracentrotus lividus sea urchin H3.3 histone variant and are probably implicated in mRNA instability. In order to identify these RNA-binding proteins, we screened a lambda-ZAPII cDNA expression library prepared from poly(A) mRNA extracted from sea urchin embryos at blastula stage. We isolated a cDNA that codes for a novel RNA-binding protein homologous to rat and human AUF1 family proteins and we refer to it as PLAUF. Proteins present in the whole lysate of the phages expressing PLAUF bound specifically in vitro the 3'UTR of the H3.3 histone transcript. Northern blot analysis revealed three PLAUF transcripts that are already present in unfertilized eggs; during development their amount increased starting from 4-blastomere embryos and reached the plateau at blastula stage. While the transcription start point was unique, longer 3'UTRs were revealed by 3'RACE approach and further cDNA library screening. Moreover RT-PCR showed the presence of at least one alternative spliced mRNA that codes for a protein with different COOH terminus. The structure of the PLAUF gene was determined by screening a P. lividus sea urchin genomic library with the PLAUF cDNA as probe. Analysis of the positive clones showed that the PLAUF gene is split in 10 exons and 9 introns spanning a distance of about 10 kb. Moreover we demonstrated that the exon 9 was alternative spliced during mRNA processing.
初步结果表明，多种蛋白质与海胆紫海胆（Paracentrotus lividus）H3.3组蛋白变体转录本的长3'非翻译区（UTR）结合，可能与mRNA的不稳定性有关。为了鉴定这些RNA结合蛋白，我们筛选了一个λ-ZAPII cDNA表达文库，该文库由囊胚期海胆胚胎中提取的poly(A) mRNA制备而成。我们分离出了一个编码与大鼠和人类AUF1家族蛋白同源的新型RNA结合蛋白的cDNA，并将其命名为PLAUF。表达PLAUF的噬菌体全裂解物中存在的蛋白质在体外特异性结合H3.3组蛋白转录本的3'UTR。Northern印迹分析显示，未受精卵中已存在三种PLAUF转录本；在发育过程中，从4细胞胚胎开始其数量增加，并在囊胚期达到稳定水平。虽然转录起始点是唯一的，但通过3'RACE方法和进一步的cDNA文库筛选发现了更长的3'UTR。此外，RT-PCR显示至少存在一种编码具有不同COOH末端蛋白质的可变剪接mRNA。通过以PLAUF cDNA为探针筛选紫海胆基因组文库，确定了PLAUF基因的结构。对阳性克隆的分析表明，PLAUF基因被分成10个外显子和9个内含子，跨度约为10 kb。此外，我们证明外显子9在mRNA加工过程中发生了可变剪接。
