Instituto Carlos Chagas, Laboratório de Regulação da Expressão Gênica, Curitiba, Paraná, Brazil.
Gene. 2010 Mar 1;452(2):72-8. doi: 10.1016/j.gene.2009.12.009. Epub 2010 Jan 7.
Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi.
在原生动物中，基因调控主要是转录后调控。mRNA 的稳定性和多核糖体的可及性被认为是受到严格调控的，这使得克氏锥虫能够在其生命周期中适应不同的环境条件。转录后调控需要 mRNA 与某些蛋白质之间的相互作用，以形成 mRNP 复合物。我们使用聚（T）珠来分离和鉴定与 poly-A+ mRNA 结合的蛋白质和蛋白质复合物，研究了蛋白质与 mRNA 之间的动态相互作用。这些级分中的蛋白质含量通过质谱（LC-MS/MS）进行分析。我们鉴定了与 mRNA 相关的 542 种 mRNP 复合物的蛋白质成分。在所有级分中都存在 24 种获得的蛋白质，而其他一些蛋白质则是特定级分所独有的：前鞭毛体多核糖体（0.37%）和后多核糖体（2.95%）级分；应激多核糖体（13.8%）和后多核糖体（40.78%）级分。鉴定出了几种已知参与 mRNA 代谢的蛋白质，这一点很重要，因为它使我们能够确认我们的 mRNP 分离方法的可靠性。该程序使我们能够首次深入了解 T. cruzi 中 mRNP 的组成和动态。
