Custódio Noélia, Antoniou Michael, Carmo-Fonseca Maria
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
Exp Cell Res. 2006 Aug 1;312(13):2557-67. doi: 10.1016/j.yexcr.2006.04.018. Epub 2006 May 9.
Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.
真核生物RNA聚合酶II是一种由12个不同亚基组成的复杂酶，在细胞中含量较低。RNA聚合酶II转录mRNA涉及该酶最大亚基羧基末端结构域（CTD）的磷酸化/去磷酸化循环。我们构建了稳定表达对α-鹅膏蕈碱具有抗性的RNA聚合酶II最大亚基（RNA Pol II LS）的小鼠细胞系。这些细胞在α-鹅膏蕈碱存在的情况下仍保持转录活性，表明外源蛋白具有功能。我们观察到，过表达的RNA Pol II LS主要处于低磷酸化状态，可溶，并以依赖CRM1的方式在细胞质中积累。我们的结果进一步表明，CTD重复序列第2位含有磷酸丝氨酸的转录活性形式的RNA Pol II LS局限于细胞核，其水平保持显著恒定。我们提出，RNA Pol II LS的核质穿梭可能提供一种机制，来控制可用于在细胞核中组装功能性酶的RNA聚合酶亚基库。
