Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.
Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.
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U1 和 U2 snRNA 的寡核苷酸靶向降解揭示了猿猴病毒 40 前 mRNA 与 snRNP 的不同相互作用。
DOI：
10.1093/nar/17.16.6553
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发表时间：
1989
影响因子：
14.9
通讯作者：
Prives,C
中科院分区：
文献类型：
--
作者：
Pan,ZQ;Ge,H;Fu,XY;Manley,JL;Prives,C
我们通过寡核苷酸靶向降解非洲爪蟾卵母细胞中的 U1 或 U2 snRNA，研究了 U1 和 U2 snRNP 颗粒在 SV40 前体 mRNA 剪接中的作用。在存在或不存在 SV40 DNA 的情况下，显微注射与 U1 或 U2 RNA 区域互补的寡核苷酸，会导致相应 snRNA 的特异性切割。出乎意料的是，当在没有引入病毒DNA或在引入病毒DNA之前注射寡核苷酸时，U1或U2 snRNA的降解要广泛得多。在共注射或预注射的卵母细胞中，这些寡核苷酸导致病毒晚期区域表达的剪接SV40 mRNA的积累显着减少，并且未剪接的晚期RNA相应增加。预注射时，两种不同的 U2 特异性寡核苷酸也抑制大肿瘤抗原和小肿瘤抗原剪接早期 mRNA 的形成。然而，即使通过预注射 U1 5' 末端特异性寡核苷酸，卵母细胞中 U1 snRNA 5' 末端发生超过 95% 的降解，也没有观察到早期前 mRNA 剪接的减少。相比之下，相同的U1 5'末端寡核苷酸，当添加到HeLa剪接提取物中时，显着抑制SV40早期前mRNA的剪接，表明U1 snRNP对于早期剪接并非完全可有可无。这些发现证实并扩展了我们之前的观察结果，即不同的前 mRNA 对 snRNP 的需求有所不同。
We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5′ end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5′ ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5′ end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 snRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.