Tombácz István, Schauer Tamás, Juhász Ildikó, Komonyi Orbán, Boros Imre
Institute of Biochemistry, Biological Research Center, Szeged, Hungary.
Gene. 2009 Oct 15;446(2):58-67. doi: 10.1016/j.gene.2009.07.012. Epub 2009 Jul 24.
The reversible phosphorylation-dephosphorylation of RNA polymerase II (Pol II) large subunit carboxyl terminal domain (CTD) during transcription cycles in eukaryotic cells generates signals for the steps of RNA synthesis and maturation. The major phosphatase specific for CTD dephosphorylation from yeast to mammals is the TFIIF-interacting CTD-phosphatase, Fcp1. We report here on the in vivo analysis of Fcp1 function in Drosophila using transgenic lines in which the phosphatase production is misregulated. Fcp1 function is essential throughout Drosophila development and ectopic up- or downregulation of fcp1 results in lethality. The fly Fcp1 binds to specific regions of the polytene chromosomes at many sites colocalized with Pol II. In accord with the strong evolutional conservation of Fcp1: (1) the Xenopus fcp1 can substitute the fly fcp1 function, (2) similarly to its S. pombe homologue, Drosophila melanogaster (Dm)Fcp1 interacts with the RPB4 subunit of Pol II, and (3) transient expression of DmFcp1 has a negative effect on transcription in mammalian cells. The in vivo experimental system described here suggests that fly Fcp1 is associated with the transcription engaged Pol II and offers versatile possibilities for studying this evolutionary conserved essential enzyme.
在真核细胞转录周期中，RNA聚合酶II（Pol II）大亚基羧基末端结构域（CTD）的可逆磷酸化-去磷酸化作用产生了RNA合成和成熟步骤所需的信号。从酵母到哺乳动物，负责CTD去磷酸化的主要磷酸酶是与TFIIF相互作用的CTD磷酸酶Fcp1。我们在此报告了利用磷酸酶产生失调的转基因品系对果蝇中Fcp1功能进行的体内分析。Fcp1功能在果蝇整个发育过程中至关重要，fcp1的异位上调或下调都会导致死亡。果蝇Fcp1与多线染色体的特定区域结合，这些区域在许多位点与Pol II共定位。鉴于Fcp1具有很强的进化保守性：（1）非洲爪蟾的fcp1可以替代果蝇fcp1的功能；（2）与粟酒裂殖酵母中的同源物类似，黑腹果蝇（Dm）Fcp1与Pol II的RPB4亚基相互作用；（3）DmFcp1的瞬时表达对哺乳动物细胞中的转录有负面影响。本文描述的体内实验系统表明，果蝇Fcp1与参与转录的Pol II相关联，并为研究这种进化保守的必需酶提供了多种可能性。
