The polyadenylation complex of Trypanosoma brucei: Characterization of the functional poly(A) polymerase

The generation of mature mRNA in the protozoan parasite Trypanosoma brucei requires coupled polyadenylation and trans splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3′ end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in T. brucei, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3′ end processing of mRNAs. Based on tandem-affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both in vivo polyadenylation and trans splicing, arguing for a general coupling of these two mRNA-processing reactions.布氏锥虫（Trypanosoma brucei，一种原生动物寄生虫）的成熟mRNA生成需要多聚腺苷酸化（polyadenylation）与反式剪接（trans splicing）的协同进行。与其他真核生物不同，我们对锥虫3'端加工机器（3′ end-processing machinery）的组成成分、作用机制及动力学过程仍知之甚少。为解析布氏锥虫多聚腺苷酸化复合物的催化核心，本研究首先鉴定出多聚腺苷酸聚合酶（poly(A) polymerase）[Tb927.7.3780]为锥虫中主要的功能性核定位酶。与之相反，另一种由含内含子基因[Tb927.3.3160]编码的多聚腺苷酸聚合酶主要定位于细胞质，且似乎无法参与mRNA的常规3'端加工过程。基于带有标签的CPSF160的串联亲和纯化（tandem-affinity purification）与质谱（mass spectrometry）分析，本研究鉴定出锥虫多聚腺苷酸化复合物的10种相关组分，包括全部4个切割多聚腺苷酸化特异性因子（CPSF）亚基、Fip1蛋白（Fip1）、切割刺激因子（CstF）50/64亚基以及Symplekin蛋白（Symplekin）的同源蛋白，另有2种假想蛋白。通过RNA干扰（RNA interference, RNAi）介导的基因敲低实验发现，多数此类因子对锥虫的生长至关重要，且同时参与体内多聚腺苷酸化与反式剪接过程，这表明这两种mRNA加工反应存在普遍的协同耦合。