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体外合成的腺病毒2信使核糖核酸前体被核提取物精确剪接。

In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts.

作者信息

Goldenberg C J

出版信息

Proc Natl Acad Sci U S A. 1984 Aug;81(15):4707-11. doi: 10.1073/pnas.81.15.4707.

DOI:10.1073/pnas.81.15.4707
PMID:6589618
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC391559/
Abstract

Precursor mRNAs were synthesized in vitro from a plasmid in which the early region 2 gene of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and used as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro splicing was accurate at the nucleotide level. The reaction occurs rapidly and without any detectable lag. The concentration of the pre-mRNA precursor during incubation appears to be an important factor for high efficiency (60%-80%) of in vitro RNA splicing. Fractionation of the splicing components as well as modifications of the DNA template to study the nucleotide-sequence requirement for in vitro splicing can now be accomplished with this system.

摘要

前体mRNA在体外由一种质粒合成，该质粒中腺病毒2的早期区域2基因与一个高效噬菌体启动子（沙门氏菌噬菌体6）融合。RNA被纯化，并用作在从MOPC - 315小鼠骨髓瘤细胞制备的核提取物存在下进行体外剪接的底物。体外剪接在核苷酸水平上是准确的。反应迅速发生且没有任何可检测到的延迟。孵育过程中前体mRNA前体的浓度似乎是体外RNA剪接高效率（60% - 80%）的一个重要因素。现在可以用这个系统完成剪接成分的分级分离以及DNA模板的修饰，以研究体外剪接的核苷酸序列要求。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/0e616499e79c/pnas00616-0102-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/ead808eca00f/pnas00616-0100-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/2fb67ea45253/pnas00616-0100-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/a3d71ffd2683/pnas00616-0100-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/14d525127261/pnas00616-0101-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/07b056e29ab4/pnas00616-0101-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/0e616499e79c/pnas00616-0102-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/ead808eca00f/pnas00616-0100-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/2fb67ea45253/pnas00616-0100-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/a3d71ffd2683/pnas00616-0100-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/14d525127261/pnas00616-0101-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/07b056e29ab4/pnas00616-0101-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eeff/391559/0e616499e79c/pnas00616-0102-a.jpg

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1
In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts.体外合成的腺病毒2信使核糖核酸前体被核提取物精确剪接。
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4707-11. doi: 10.1073/pnas.81.15.4707.
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In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.腺病毒2型基因组早期区域2所指定的纯化前体RNA的体外剪接
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Accurate and efficient in vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.腺病毒2型基因组早期区域2所指定的纯化前体RNA的准确且高效的体外剪接。
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引用本文的文献

1
Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro.体外mRNA剪接所需的来自HeLa细胞的两个组分的分离与特性鉴定。
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2
Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones.合成寡核苷酸尾抑制玉米醇溶蛋白cDNA克隆的SP6转录本的体外和体内翻译。
Nucleic Acids Res. 1986 Feb 11;14(3):1511-24. doi: 10.1093/nar/14.3.1511.
3
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本文引用的文献

1
Speculations on RNA splicing.关于RNA剪接的推测。
Cell. 1981 Mar;23(3):643-6. doi: 10.1016/0092-8674(81)90425-6.
2
A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.β0地中海贫血基因剪接位点的单碱基变化导致异常的RNA剪接。
Cell. 1982 Jul;29(3):903-11. doi: 10.1016/0092-8674(82)90452-4.
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A catalogue of splice junction sequences.剪接连接序列目录。
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Nucleic Acids Res. 1987 Nov 25;15(22):9279-98. doi: 10.1093/nar/15.22.9279.
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Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.在体外剪接反应中，将小鼠胰岛素信使核糖核酸前体组装成60 - 40S核糖核蛋白复合体。
Nucleic Acids Res. 1986 May 12;14(9):3687-701. doi: 10.1093/nar/14.9.3687.
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Antisense RNA inhibits splicing of pre-mRNA in vitro.反义RNA在体外抑制前体mRNA的剪接。
EMBO J. 1988 Aug;7(8):2523-32. doi: 10.1002/j.1460-2075.1988.tb03100.x.
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The quantitation and distribution of splicing intermediates in HeLa cells and adenovirus RNAs.HeLa细胞和腺病毒RNA中剪接中间体的定量与分布
Nucleic Acids Res. 1987 Sep 11;15(17):7103-24. doi: 10.1093/nar/15.17.7103.
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In vivo splicing of the premRNAs from early region 3 of adenovirus-2: the products of cleavage at the 5' splice site of the common intron.腺病毒2型早期区域3前体mRNA的体内剪接：共同内含子5'剪接位点的切割产物
Nucleic Acids Res. 1986 Feb 11;14(3):1187-207. doi: 10.1093/nar/14.3.1187.
Nucleic Acids Res. 1982 Jan 22;10(2):459-72. doi: 10.1093/nar/10.2.459.
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Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site.通过在mRNA剪接位点进行位点特异性诱变来解析重叠病毒基因的功能。
Nature. 1982 Feb 4;295(5848):380-4. doi: 10.1038/295380a0.
5
Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme.噬菌体SP6特异性RNA聚合酶。I. 酶的分离与特性鉴定。
J Biol Chem. 1982 May 25;257(10):5772-8.
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Nuclear RNA is spliced in the absence of poly(A) addition.核RNA在没有添加聚腺苷酸的情况下进行剪接。
Cell. 1981 Oct;26(1 Pt 1):39-46. doi: 10.1016/0092-8674(81)90031-3.
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In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.腺病毒2型基因组早期区域2所指定的纯化前体RNA的体外剪接
Proc Natl Acad Sci U S A. 1981 Sep;78(9):5430-4. doi: 10.1073/pnas.78.9.5430.
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Transcription and processing of adenoviral RNA by extracts from HeLa cells.用HeLa细胞提取物对腺病毒RNA进行转录和加工
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4092-6. doi: 10.1073/pnas.78.7.4092.
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Beta + thalassemia: aberrant splicing results from a single point mutation in an intron.β+地中海贫血：异常剪接由内含子中的单个点突变引起。
Cell. 1981 Dec;27(2 Pt 1):289-98. doi: 10.1016/0092-8674(81)90412-8.
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Organization and expression of eucaryotic split genes coding for proteins.编码蛋白质的真核生物断裂基因的组织与表达。
Annu Rev Biochem. 1981;50:349-83. doi: 10.1146/annurev.bi.50.070181.002025.