Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi
Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi
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DOI：
10.1111/j.1742-4658.2010.07747.x
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发表时间：
2010-08-01
期刊：
影响因子：
5.4
通讯作者：
Goldenberg, Samuel
中科院分区：
文献类型：
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作者：
Holetz, Fabiola B.;Alves, Lysangela R.;Goldenberg, Samuel
在锥虫中，基因表达的调节主要发生在转录后水平。先前的研究已经揭示了克氏锥虫细胞质中的非翻译mRNA。在此之前，我们已经确定并克隆了TcDHH1蛋白，一个死亡盒RNA解旋酶。Dhh1在多种真核生物中参与多种RNA相关过程。据报道，它还在酵母、动物细胞、布氏锥虫和T.克鲁兹TcDHH1定位于离散的细胞质病灶，其根据生命周期状态和营养条件而变化。为了研究含有TcDHH1的mRNP的组成，我们使用外鞭毛体裂解物用抗TcDHH1进行免疫沉淀测定。通过MS测定mRNP的蛋白质含量，并使用免疫前血清作为对照。我们还进行了核糖组学的方法，以确定存在于TcDHH1免疫沉淀复合物的mRNA。为了这个目的，竞争性微阵列杂交对阴性对照，非沉淀部分进行。我们的研究结果表明，与TcDHH1相关的mRNA在上鞭毛体阶段主要在T.克鲁兹生命周期这些数据表明，含有TcDHH1的mRNP参与mRNA代谢，调节至少上鞭毛体特异性基因的表达。
In trypanosomatids, the regulation of gene expression occurs mainly at the post-transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA-related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti-TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote-specific genes.