Gupta S, Busch R K, Singh R, Reddy R
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1990 Nov 5;265(31):19137-42.
The cap structure in human U6 small nuclear (sn)RNA, gamma-monomethylguanosine triphosphate (meGTP), was conjugated to human serum albumin and used as antigen to raise polyclonal antibodies in rabbits. The resulting antibodies reacted specifically with meGTP but not with GTP, GDP, GMP, meGMP, meATP, meCTP, meUTP, or with methyl phosphate in enzyme-linked immunosorbent assay and/or in radioimmunoassays. Although less efficiently, meGDP was also recognized by these antibodies. Indirect immunofluorescence studies with anti-meGTP antibodies showed predominantly nuclear immunofluorescence. Anti-meGTP antibodies immunoprecipitated intact U6 snRNA from a mixture of HeLa cell RNAs. In addition to the U6 snRNA, anti-meGTP antibodies immunoprecipitated several additional small RNAs that varied in length from approximately 50 to 330 nucleotides. These RNAs contained the meGTP cap structure and are structurally distinct from U6 snRNA. One of these meGTP-containing RNAs was found to be previously characterized 7SK RNA; human 7SK RNA synthesized in vitro also contained the same cap structure. Results obtained in this study provide evidence for the presence of gamma-monomethyl-GTP cap structure in a wide spectrum of human cellular RNAs. These antibodies will be useful in studying the structure and function of this new family of small RNAs.
人U6小核（sn）RNA中的帽结构，γ-单甲基鸟苷三磷酸（meGTP），与人类血清白蛋白偶联，并用作抗原在兔体内产生多克隆抗体。所得抗体在酶联免疫吸附测定和/或放射免疫测定中与meGTP特异性反应，但不与GTP、GDP、GMP、meGMP、meATP、meCTP、meUTP或甲基磷酸反应。尽管效率较低，但这些抗体也能识别meGDP。用抗meGTP抗体进行的间接免疫荧光研究显示主要为核免疫荧光。抗meGTP抗体从HeLa细胞RNA混合物中免疫沉淀完整的U6 snRNA。除了U6 snRNA，抗meGTP抗体还免疫沉淀了几种长度约为50至330个核苷酸的其他小RNA。这些RNA含有meGTP帽结构，在结构上与U6 snRNA不同。其中一种含meGTP的RNA被发现是先前已鉴定的7SK RNA；体外合成的人7SK RNA也含有相同的帽结构。本研究获得的结果为广泛的人类细胞RNA中存在γ-单甲基-GTP帽结构提供了证据。这些抗体将有助于研究这个新的小RNA家族的结构和功能。
