Moon Kyong Hwa, Zhao Xinliang, Yu Yi-Tao
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA.
Methods Mol Biol. 2006;322:149-63. doi: 10.1007/978-1-59745-000-3_11.
Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing.
非洲爪蟾卵母细胞已在许多实验室中作为一种实验系统，用于研究各种生物学过程。在此，我们描述其在信使核糖核酸前体（pre-mRNA）剪接过程中对剪接体小核核糖核酸（snRNA）功能研究的应用，该过程在非洲爪蟾卵母细胞中极其高效地发生。将与感兴趣的snRNA互补的DNA寡核苷酸注入卵母细胞细胞质中。该寡核苷酸随后扩散到细胞核中并与靶标snRNA杂交，从而通过内源性核糖核酸酶H活性触发snRNA降解。到内源性snRNA被耗尽时，DNA寡核苷酸本身会被内源性脱氧核糖核酸酶（DNase）活性降解。原则上，此程序能使人们定量耗尽任何选定的snRNA。随后，可将体外构建的拯救性snRNA注入snRNA耗尽的卵母细胞中以恢复剪接功能。重建后，将放射性标记的剪接底物注入卵母细胞核中。然后手动分离这些卵母细胞核，并用于制备用于剪接测定的核RNA和用于剪接体组装测定的核提取物。因此，可以测试注入的拯救性snRNA重建剪接的能力。由于所有类型的拯救性snRNA（例如，突变snRNA、带有或不带有修饰核苷酸的snRNA）都可以很容易地构建，从该程序获得的结果为感兴趣的特定snRNA在pre-mRNA剪接中的功能提供了有价值的信息。
