Echeverria M, Lahmy S
Laboratoire de Physiologie et Biologie Moléculaire Végétale, Université de Perpignan, URA CNRS 565, France.
Nucleic Acids Res. 1995 Dec 25;23(24):4963-70. doi: 10.1093/nar/23.24.4963.
In radish pre-rRNA primary processing cleavage occurs at a UUUUCGCGC element (motif P) mapped in the 5'-external transcribed spacer (Delcasso-Tremousaygue et al., 1988). Significantly, motif P is part of a cluster of homologous elements including three UUUUCCGG elements (motifs A123) and a single UUUUGCCCC element (motif B). Here we used the EMSA to identify in radish extracts an RNA-binding activity, NF C, that specifically interacts with the pre-rRNA A123BP sequence. Using different RNA probes and competitors we show that NF C recognises a 38 base RNA sequence including the 3'-end of motif A3 and motifs B and P. NF C binds to poly U, but not to poly A, poly C or poly G. Therefore we used poly (U) Sepharose chromatography as a final step to obtain pure NF C fractions. These, analysed by SDS-PAGE, revealed two major polypeptides of 67 and 60 kDa. According to UV cross-linking analysis the 67 kDa polypeptide corresponds to NF C activity, while the 60 kDa species is a proteolysed form of this protein. We also showed that NF C is enriched in nuclear extracts. Based on its stringent RNA substrate specificity and its nuclear localisation we propose that NF C is involved in pre-rRNA primary processing in plants.
在萝卜中，前体rRNA的初级加工切割发生在5'-外部转录间隔区中定位的UUUUCGCGC元件（基序P）处（德尔卡索 - 特雷穆赛亚盖等人，1988年）。值得注意的是，基序P是一组同源元件的一部分，包括三个UUUUCCGG元件（基序A123）和一个单一的UUUUGCCCC元件（基序B）。在这里，我们使用电泳迁移率变动分析（EMSA）在萝卜提取物中鉴定出一种RNA结合活性，即NF C，它与前体rRNA的A123BP序列特异性相互作用。使用不同的RNA探针和竞争物，我们表明NF C识别一个38个碱基的RNA序列，包括基序A3的3'-末端以及基序B和P。NF C与聚U结合，但不与聚A、聚C或聚G结合。因此，我们使用聚（U）琼脂糖凝胶柱层析作为最后一步来获得纯的NF C组分。通过SDS-PAGE分析，这些组分显示出两条主要的多肽，分子量分别为67 kDa和60 kDa。根据紫外线交联分析，67 kDa的多肽对应于NF C活性，而60 kDa的物种是该蛋白质的一种蛋白水解形式。我们还表明NF C在核提取物中富集。基于其严格的RNA底物特异性及其核定位，我们提出NF C参与植物前体rRNA的初级加工。
