Brooks S A, Rigby W F
Department of Medicine, Dartmouth Medical School, Hanover, NH 03756, USA.
Nucleic Acids Res. 2000 May 15;28(10):E49. doi: 10.1093/nar/28.10.e49.
Post-transcriptional regulation is an important mechanism in cellular response to stimuli, allowing for the rapid and discrete expression of relevant proteins. Genes regulated by this mechanism have specific cis -acting elements, frequently in their 3' untranslated regions (UTRs), that have been shown to serve as recognition sites for trans -acting RNA-binding proteins. Unfortunately, the identification of specific mRNA ligands for different RNA binding proteins in vivo has been limited by a lack of adequate methodology. We have developed a novel technique that addresses this shortcoming, using immunoprecipitation of RNA binding proteins from polysomes followed by RT-PCR and library screening to identify the in vivo mRNA ligands of RNA binding proteins. Utilizing this approach, we have identified 32 known and 16 novel mRNAs specifically bound by the heterogeneous nuclear ribonucleoprotein (hnRNP) A2. Of the clones identified, 74% contained AU-rich elements and/or poly-uridine tracts in their 3' UTRs, cis -acting elements that have been established as impacting mRNA stability. The high percentage of clones containing these uridine-rich sequences compares favorably with the high affinity binding of poly-uridine RNA by hnRNP A2 in vitro. These data thus support the representative nature of the technique.
转录后调控是细胞对刺激作出反应的一种重要机制，可实现相关蛋白质的快速且特异性表达。受该机制调控的基因具有特定的顺式作用元件，这些元件常位于其3'非翻译区（UTR），已被证明可作为反式作用RNA结合蛋白的识别位点。遗憾的是，由于缺乏足够的方法，在体内鉴定不同RNA结合蛋白的特异性mRNA配体受到了限制。我们开发了一种新技术来解决这一缺点，即从多核糖体中免疫沉淀RNA结合蛋白，随后进行逆转录聚合酶链反应（RT-PCR）和文库筛选，以鉴定RNA结合蛋白在体内的mRNA配体。利用这种方法，我们鉴定出了32种已知的和16种新的mRNA，它们特异性地与不均一核核糖核蛋白（hnRNP）A2结合。在鉴定出的克隆中，74%在其3'UTR中含有富含AU的元件和/或多聚尿苷序列，这些顺式作用元件已被确定会影响mRNA的稳定性。含有这些富含尿苷序列的克隆比例很高，这与hnRNP A2在体外对多聚尿苷RNA的高亲和力结合情况相符。因此，这些数据支持了该技术的代表性。
