Bhattacharya S, Giordano T, Brewer G, Malter J S
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792, USA.
Nucleic Acids Res. 1999 Mar 15;27(6):1464-72. doi: 10.1093/nar/27.6.1464.
Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.
细胞激活与转录和转录后水平上基因表达的多种广泛变化相关。AUF1是一种最近被描述的细胞质蛋白，它可能参与富含AU（ARE）的mRNA的转录后调控（PTR），包括那些编码细胞因子和原癌基因的mRNA。如果存在AREs，或者所讨论的mRNA在体外或体内与AUF1相互作用，那么受AUF1介导的PTR作用的单个mRNA是可以预测的。然而，几乎没有（如果有的话）用于表征受AUF1进行PTR作用的mRNA总体库的通用方法。为了鉴定这些mRNA，我们在体外将来自丝裂原激活的外周血单核细胞（PBMCs）的总mRNA与AUF1一起孵育。AUF1 - mRNA复合物在膜上迁移受阻，结合的mRNA用高盐洗脱，然后要么用于构建cDNA文库，要么在洗脱和构建cDNA文库之前再次或第三次与AUF1结合。我们从130个克隆中获得了部分核苷酸序列，这表明AUF1选择的文库富含含有3'非翻译区AREs的mRNA，包括大量早期反应基因cDNA。作为对该方法有效性的测试，我们还表明文库中随机选择的一种新的mRNA在细胞激活后会被稳定化。
