Neugebauer K M, Stolk J A, Roth M B
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
J Cell Biol. 1995 May;129(4):899-908. doi: 10.1083/jcb.129.4.899.
The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.
真核生物前体mRNA中内含子的去除发生在一种称为剪接体的大型核糖核蛋白复合物中。我们针对六种SR蛋白家族中的四种产生了一种单克隆抗体（mAb 16H3），这四种SR蛋白是已知的剪接位点选择和剪接体组装的调节因子。除了反应性SR蛋白SRp20、SRp40、SRp55和SRp75外，mAb 16H3还能与人、青蛙和果蝇提取物中大约20种不同的核蛋白结合，而酵母未检测到可表达该表位。这些抗原显示为核内、非核仁的，并集中在RNA聚合酶II转录的活性位点，这表明它们参与前体mRNA的加工。实际上，在核提取物中观察到的大多数反应性蛋白在体外组装的剪接体（E和/或B复合物）中也能检测到，包括U1小核核糖核蛋白颗粒的U1 70K组分以及U2AF的两个亚基。有趣的是，16H3表位被定位到一个由40个氨基酸组成的多肽上，该多肽几乎完全由精氨酸与谷氨酸和天冬氨酸交替组成。所有已鉴定的抗原，包括酵母Prp22的人类同源物（HRH1），都含有一个类似的结构元件，其特征是精氨酸与丝氨酸、谷氨酸和/或天冬氨酸交替出现。这些结果表明，更多的剪接体组分含有这种富含精氨酸的结构域。由于它在后生动物中是保守的，我们提出mAb 16H3识别的“交替精氨酸”结构域可能代表前体mRNA剪接因子的一个共同功能元件。
