3′ end processing of Drosophila melanogaster histone Pre-mRNAs:: Requirement for phosphorylated drosophila stem-loop binding protein and coevolution of the histone Pre-mRNA processing system
3′ end processing of Drosophila melanogaster histone Pre-mRNAs:: Requirement for phosphorylated drosophila stem-loop binding protein and coevolution of the histone Pre-mRNA processing system
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DOI：
10.1128/mcb.22.18.6648-6660.2002
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发表时间：
2002-09-01
影响因子：
5.3
通讯作者：
Marzluff, WF
中科院分区：
文献类型：
--
作者：
Dominski, Z;Yang, XC;Marzluff, WF
含有由果蝇组蛋白基因编码的加工信号的人工合成的Pre-mRNAs在果蝇培养细胞和0-13 h龄胚胎的核提取液中经历了有效而忠实的内切。体外切割的生化要求类似于先前描述的哺乳动物组蛋白前mRNAs的3‘端加工。果蝇3‘末端加工不需要ATP，在EDTA存在下发生。然而，与哺乳动物的加工相反，果蝇的加工在茎环之后产生结束四个核苷酸的最终产品。通过耗尽果蝇茎环结合蛋白(DSLBP)的提取物，果蝇底物的切割被取消，这表明dSLBP和组蛋白Pre-mRNA中的茎环结构都是加工机械的重要组成部分。利用杆状病毒系统在昆虫细胞中表达的重组dSLBP有效地补充了耗尽的提取物。只有RNA结合区加上dSLBP C末端的17个氨基酸才能进行加工。在昆虫细胞中表达的全长dSLBP在C-末端的四个残基上被定量磷酸化。重组dSLBP的去磷酸化降低了加工活性。人和果蝇的SLBP是不可互换的，并且强烈地抑制异源提取物中的加工。在哺乳动物提取物中处理组蛋白前mRNA时，dSLBP的RNA结合域不能取代人SLBP的RNA结合域。除了茎环结构和dSLBP外，果蝇核提取物中3‘的加工还取决于果蝇核提取物中是否存在位于茎下游约20个核苷酸的一小段嘌呤，以及一个Sm反应因子，很可能是脊椎动物U7 SnRNP的果蝇对应物。
Synthetic pre-mRNAs containing the processing signals encoded by Drosophila melanogaster histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3' end processing of mammalian histone pre-mRNAs. Drosophila 3' end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3' processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP.