Dominski Zbigniew, Yang Xiao-Cui, Raska Christy S, Santiago Carlos, Borchers Christoph H, Duronio Robert J, Marzluff William F
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 27599, USA.
Mol Cell Biol. 2002 Sep;22(18):6648-60. doi: 10.1128/MCB.22.18.6648-6660.2002.
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.
含有由黑腹果蝇组蛋白基因编码的加工信号的合成前体mRNA，在从果蝇培养细胞和0至13小时龄胚胎制备的核提取物中能高效且准确地进行内切核酸酶切割。体外切割的生化要求与先前描述的哺乳动物组蛋白前体mRNA的3'末端加工的要求相似。果蝇的3'末端加工不需要ATP，且在EDTA存在的情况下发生。然而，与哺乳动物的加工不同，果蝇的加工产生的最终产物在茎环后四个核苷酸处结束。通过耗尽果蝇茎环结合蛋白（dSLBP）提取物，果蝇底物的切割被消除，这表明dSLBP和组蛋白前体mRNA中的茎环结构都是加工机制的重要组成部分。利用杆状病毒系统在昆虫细胞中表达的重组dSLBP能有效地补充耗尽的提取物。加工仅需要dSLBP的RNA结合结构域加上C末端的17个氨基酸。在昆虫细胞中表达的全长dSLBP在C末端区域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不可互换，并且在异源提取物中强烈抑制加工。在哺乳动物提取物中，dSLBP的RNA结合结构域不能替代人SLBP的RNA结合结构域用于组蛋白前体mRNA的加工。除了茎环结构和dSLBP外，果蝇核提取物中的3'加工还依赖于位于茎下游约20个核苷酸处的一小段嘌呤的存在，以及一种Sm反应因子，很可能是脊椎动物U7 snRNP的果蝇对应物。
