Keller W, Bienroth S, Lang K M, Christofori G
Department of Cell Biology, University of Basel, Switzerland.
EMBO J. 1991 Dec;10(13):4241-9. doi: 10.1002/j.1460-2075.1991.tb05002.x.
Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.
切割与聚腺苷酸化因子（CPF）在信使RNA前体的3'加工过程中，对于切割以及随后的聚腺苷酸化反应都是必需的。在此，我们研究了CPF和聚（A）聚合酶与短RNA底物的相互作用。CPF激活聚（A）聚合酶，使其延伸携带典型六聚体识别信号AAUAAA的RNA引物。凝胶迁移率变动分析和竞争实验表明，CPF能特异性结合此类RNA。CPF结合后，通过紫外线照射，35 kDa和160 kDa的两种多肽可与RNA共价交联。这些多肽可能分别对应于纯化的CPF组分中最小和最大的亚基。此外，化学修饰排除实验表明，CPF直接与RNA中的AAUAAA识别信号相互作用。整个六聚体信号都参与CPF的结合，因为其任何一个碱基的修饰都会干扰复合物的形成。
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