Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA.
Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA.
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对剪接提取物中腺病毒 2 主要晚期前体 mRNA 的 RNase-A 抗性区域的分析揭示了核成分与底物 RNA 的有序相互作用。
DOI：
10.1016/0022-2836(87)90032-5
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发表时间：
1987
影响因子：
5.6
通讯作者：
A. Krämer
中科院分区：
文献类型：
--
作者：
A. Krämer
采用RNase A保护实验研究了HeLa细胞剪接提取物中腺病毒2主要晚期转录单位衍生的前体mrna与核成分的相互作用。在提取物中孵育后，前体rna中的两个区域对RNase a的消化产生抗性。在30°C的短孵育时间（5分钟）后，获得了从内含子分支点上游定位的片段。10分钟或更长时间后，来自5 '剪接位点的额外寡核苷酸被保护。RNase A对不同RNA底物的保护表明，组分与分支点区域的结合不需要5 '剪接位点。为了与该位点相互作用，3 '剪接位点上游的聚嘧啶延伸是必不可少的。与5 ‘剪接位点的结合只发生在内含子完整的3 ’端存在的情况下。将提取物与仅含有3 ‘剪接位点的过量未标记RNA进行预孵育，可导致分支点区域和5 ’剪接位点的有效结合竞争，而仅含有5 '剪接位点序列的RNA对mRNA前体的相互作用没有影响。这表明，与5 '剪接位点的稳定结合需要事先结合分支点区域的组分。当剪接复合物被RNase A酶切时，很明显35s复合物中只有分支点区域被隔离成核糖核蛋白（RNP）结构。只有在组装了50s剪接复合体后，5 '剪接位点才会对RNase A产生抗性。利用互补的寡脱氧核苷酸和RNase H降解U1、U2和U4 RNA中的特定区域来分析U小核RNPs （snRNPs）参与保护反应。U2 RNA的5′端对分支点区域的保护至关重要。U2 RNA环中的RNA序列（核苷酸65至78）是在5 '剪接位点形成抗RNA - a结构所必需的。综上所述，这些结果表明U2 snRNP参与了预剪接复合物的形成，其RNA的5 '端参与了观察到的结合。Ul和U4/U6 snRNPs结合后可转化为50s剪接复合体，这也需要U2 RNA环中的序列。讨论了单个snRNPs之间以及snRNPs与前体mrna之间可能的相互作用。
An RNase A protection assay was employed to investigate the interaction of nuclear components with a precursor-mRNA derived from the adenovirus 2 major late transcription unit in a splicing extract from HeLa cells. Upon incubation in the extract, two regions in the precursor-RNA become resistant to digestion with RNase A. After short incubation times (5 min) at 30 °C, fragments mapping upstream from the branch point in the intron are obtained. After ten minutes or more, additional oligonucleotides, derived from the 5′ splice site, are protected. RNase A protection of different RNA substrates demonstrates that a 5′ splice site is not required for the binding of components to the branch point region. For interaction with this site, the polypyrimidine stretch just upstream from the 3′ splice site is essential. Binding to the 5′ splice site occurs only in the presence of an intact 3′ end of the intron. Preincubation of the extract with excess unlabelled RNA containing only a 3′ splice site leads to efficient competition of binding, both in the branch point region and at the 5′ splice site, whereas an RNA that contains only 5′-splice-site sequences has no effect on the interaction with the mRNA precursor. This indicates that stable association with the 5′ splice site requires prior binding of components in the branch point region. When splicing complexes are digested with RNase A, it becomes apparent that only the branch point region is sequestered into a ribonucleoprotein (RNP) structure in the 35 S complex. The 5′ splice site becomes resistant to RNase A only when a 50 S splicing complex has been assembled.Degradation of specific regions in U1, U2 and U4 RNA with complementary oligodeoxynucleotides and RNase H has been used to analyse involvement of the U small nuclear RNPs (snRNPs) in the protection reaction. The 5′ end of U2 RNA is essential for protection of the branch point region. RNA sequences in a loop of U2 RNA (nucleotides 65 to 78) are required for the formation of an RNase-A-resistant structure at the 5′ splice site. Taken together, these results suggest that U2 snRNP participates in the formation of a pre-splicing complex, the 5′ end of its RNA being involved in the observed binding. Conversion to a 50 S splicing complex is obtained after the binding of Ul and U4/U6 snRNPs, which also requires sequences in a loop of U2 RNA. Possible interactions between the individual snRNPs and between snRNPs and precursor-mRNA are discussed.
保守内含子序列的突变影响酵母剪接途径中的多个步骤，特别是剪接体的组装。
DOI：
10.1002/j.1460-2075.1986.tb04412.x
发表时间：
1986
期刊：
The EMBO journal
影响因子：
--
作者：
Vijayraghavan,U;Parker,R;Tamm,J;Iimura,Y;Rossi,J;Abelson,J;Guthrie,C
通讯作者：
Guthrie,C
在体外剪接反应中将小鼠胰岛素信使 RNA 前体组装成 60-40S 核糖核蛋白复合物。
影响因子：
14.9
作者：
Kaltwasser,G;Spitzer,SG;Goldenberg,CJ
通讯作者：
Goldenberg,CJ