MESSENGER-RNA POLYADENYLATE-BINDING PROTEIN - GENE ISOLATION AND SEQUENCING AND IDENTIFICATION OF A RIBONUCLEOPROTEIN CONSENSUS SEQUENCE
MESSENGER-RNA POLYADENYLATE-BINDING PROTEIN - GENE ISOLATION AND SEQUENCING AND IDENTIFICATION OF A RIBONUCLEOPROTEIN CONSENSUS SEQUENCE
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DOI：
10.1128/mcb.6.8.2932
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发表时间：
1986-08-01
影响因子：
5.3
通讯作者：
DREYFUSS, G
中科院分区：
文献类型：
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作者：
ADAM, SA;NAKAGAWA, T;DREYFUSS, G
我们鉴定并制备了与酿酒酵母中poly（A）+RNA相互作用的主要蛋白质的抗体。在完整的酵母细胞中，通过紫外光与poly（A）+ RNA交联的主要蛋白质具有72，000、60，000和50，000的表观分子量。RNA的poly（A）片段选择性地与72，000分子量的蛋白质（72 K蛋白）交联。用纯化的UV-交联的RNA-蛋白（RNP）复合物免疫的小鼠产生针对三种主要RNP蛋白的抗体。用抗RNP血清筛选构建在酵母gt 11表达载体中的酵母基因组DNA文库，并分离重组噬菌体克隆。一个携带2.5-丝氨酸蛋白酶插入物的重组噬菌体YPA72.1产生了一个大的β-丝氨酸蛋白酶。半乳糖苷酶-RNP融合蛋白来自该融合蛋白上的抗RNP血清的亲和力选择的抗体识别与完整细胞中的RNA的poly（A）区段交联的单个72 K蛋白。 此外，YPA72.1的融合蛋白具有特异性poly（A）结合活性。因此，YPA72.1编码72 K poly（A）-结合蛋白。免疫荧光显微镜显示该蛋白定位于细胞质中。在体外翻译的杂交选择的mRNA产生的72 K聚（A）结合蛋白，和mRNA印迹分析检测到一个单一的2.1-腺苷酸酶mRNA。DNA印迹分析表明poly（A）结合蛋白的单一基因。DNA序列分析的基因组克隆跨越整个基因揭示了一个长的开放阅读框编码64,272分子量的蛋白质与几个不同的结构域和重复的结构元件。11至13个氨基酸的序列在这种蛋白质中重复三次。引人注目的是，这个重复序列（RNP共有序列）是高度同源的一个序列，是重复两次在一个主要的哺乳动物异质核RNP蛋白，A1。重复RNP一致序列的保守性表明信使RNP和异质核RNP蛋白具有重要的功能和共同的进化起源。
We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the .lambda.gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, .lambda.YPA72.1, bearing a 2.5-kilobase insert, produced a large .beta.-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of .lambda.YPA72.1 had specific poly(A)-binding activity. Therefore, .lambda.YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.