Multiple factors are required for poly(A) addition to a mRNA 3' end.
Multiple factors are required for poly(A) addition to a mRNA 3' end.
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将 Poly(A) 添加到 mRNA 3 末端需要多种因素。
DOI：
10.1101/gad.2.5.588
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发表时间：
1988
影响因子：
10.5
通讯作者：
Nevins,JR
中科院分区：
文献类型：
--
作者：
McDevitt,MA;Gilmartin,GM;Reeves,WH;Nevins,JR
细胞核中前 mRNA 的多腺苷酸化涉及特定的核酸内切裂解，然后添加大约 200 个腺苷酸残基。我们检测了 HeLa 核提取物的催化聚腺苷酸加成反应的活性。通过体外添加的聚腺苷酸长度大约为 200 个核苷酸的观察结果表明了体外测定的真实性。我们对核提取物进行了分级，以定义参与特定聚 (A) 添加的成分。 HeLa 核提取物的 DEAE-Sephacel 色谱中没有任何一个组分具有特定的聚 (A) 加成活性。然而，如果将各个部分重新组合，活性就会恢复，表明存在多种成分。进一步的分级显示存在至少两个聚（A）加成反应所需的因子。重构的系统保留了粗提取物中所见的特征和特异性。对其中一个因子的额外纯化强烈表明它是一种先前表征的聚腺苷酸聚合酶，当在不存在其他因子的情况下进行分析时，可以将 AMP 添加到 RNA 末端，但没有特异性。因此，反应的其他组分可以为该过程提供特异性。与 3' 裂解反应相反，通过微球菌核酸酶消化测定，poly(A) 加成机制不具有必需的 RNA 成分，抗 Sm 血清也不会抑制该反应。因此，多腺苷酸化 mRNA 3' 末端形成的整个过程是复杂的，需要不同核成分的协同作用。
Polyadenylation of pre-mRNAs in the nucleus involves a specific endonucleolytic cleavage, followed by the addition of approximately 200 adenylic acid residues. We have assayed HeLa nuclear extracts for the activity that catalyzes the poly(A) addition reaction. The authenticity of the in vitro assay was indicated by the observation that the poly(A) tract added in vitro is approximately 200 nucleotides in length. We have fractionated nuclear extracts in order to define components involved in specific poly(A) addition. No single fraction from DEAE-Sephacel chromatography of a HeLa nuclear extract possessed the specific poly(A) addition activity. However, if the various fractions were recombined, activity was restored, indicating the presence of multiple components. Further fractionation revealed the presence of at least two factors necessary for the poly(A) addition reaction. The reconstituted system retains the characteristics and specificity seen in the crude extract. Additional purification of one of the factors strongly suggests it to be a previously characterized poly(A) polymerase which, when assayed in the absence of the other factor, can add AMP to an RNA terminus but without specificity. Thus, the other component of the reaction may provide specificity to the process. In contrast to the 3' cleavage reaction, the poly(A) addition machinery does not possess an essential RNA component, as assayed by micrococcal nuclease digestion, nor do anti-Sm sera inhibit the reaction. Thus, the total process of formation of a polyadenylated mRNA 3' end is complex and requires the concerted action of distinct nuclear components.