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首页> 外文期刊>Molecular and Cellular Biology >Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA
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Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA

机译：茎环结合蛋白通过稳定U7 snRNP与组蛋白前mRNA的结合来促进3'-末端形成

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摘要

The 3′ end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3′-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3′ processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.
机译：组蛋白mRNA的3'端是在保守的茎环序列之后，通过内转录裂解初级转录本而形成的。裂解反应需要至少两个 trans 作用因子：与茎环序列结合的茎环结合蛋白（SLBP），以及与裂解下游序列相互作用的U7 snRNP现场。从核提取物中去除SLBP取消了3'末端加工，重组SLBP的添加恢复了耗尽提取物的加工活性。为了确定3′加工所必需的人SLBP区域，测试了蛋白质的各种缺失突变体与SLBP耗尽提取物互补的能力。人SLBP的整个N末端结构域和大部分C末端结构域对于加工都是必不可少的。有效支持组蛋白pre-mRNA裂解的最小蛋白质由包含73个氨基酸的RNA结合结构域的93个氨基酸和紧邻其C末端的20个氨基酸组成。在全长SLBP的背景下，用无关序列替换这20个残基可减少> 90％的处理。用抗SLBP抗体进行的共免疫沉淀实验表明，只有在含有正确定位的U7 snRNP结合位点的前mRNA底物存在时，SLBP和U7 snRNP才能形成稳定的复合物。 SLBP的作用之一是稳定组蛋白前体mRNA与U7 snRNP的相互作用。

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