A novel essential RNA-binding protein complex in Trypanosoma brucei

ZC3H5 is an essential cytoplasmic trypanosome protein with a single Cx7Cx5Cx3H zinc finger domain. We here show that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. ZC3H5 interacts directly with Tb927.11.4900, which in turn interacts with Tb927.7.3040. Tb927.11.4900 has a circularly permuted GTPase domain, which is required for the Tb927.7.3040 interaction. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which were enriched in the motif (U/A)UAG(U/A). Tethering of ZC3H5, or other complex components, to a reporter repressed its expression. However, depletion of ZC3H5 in vivo did not increase the abundance of ZC3H5-bound mRNAs; instead, counter-intuitively, there were very minor decreases in a few targets. This was accompanied by marked increases in the abundance of very stable mRNAs encoding ribosomal proteins. Depletion also resulted in an increase in monosomes at the expense of large polysomes, and appearance of a "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex is implicated in quality control during the translation of sub-optimal open reading frames, and that its assembly is regulated by GTP hydrolysis and GTP-GDP exchange.

ZC3H5是锥虫中一种必需的胞质蛋白，携带有单个Cx7Cx5Cx3H型锌指结构域（zinc finger domain）。本研究证实，ZC3H5与另外三种蛋白形成复合物，这三种蛋白分别由基因Tb927.11.4900、Tb927.8.1500及Tb927.7.3040编码。ZC3H5可直接与Tb927.11.4900发生相互作用，而Tb927.11.4900又可与Tb927.7.3040产生结合。Tb927.11.4900带有一个环状重排GTP酶结构域（GTPase domain），该结构域是其与Tb927.7.3040相互作用的必要条件。RNA免疫沉淀（RNA immunoprecipitation）实验结果显示，ZC3H5优先结合翻译效率低下、稳定性较差的mRNA，这类mRNA的5'非翻译区（5'-untranslated regions）及编码区中，(U/A)UAG(U/A)基序的富集程度显著升高。将ZC3H5或该复合物的其他组分锚定至报告基因后，可抑制报告基因的表达。然而，在体内敲低ZC3H5并未使ZC3H5结合的mRNA丰度提升；反而出乎意料的是，少数靶标mRNA的丰度仅出现极轻微的下降。这一变化同时伴随着编码核糖体蛋白的高度稳定mRNA的丰度显著升高。敲低ZC3H5还会导致单核糖体增多，大型多核糖体占比随之下降，并出现含有两个80S亚基与一个40S亚基的半体二聚核糖体（halfmer disomes）结构。我们推测，ZC3H5复合物参与了亚最优开放阅读框（open reading frame, ORF）翻译过程中的质量控制，且其组装过程受GTP水解与GTP-GDP交换的调控。