Acestor Nathalie, Panigrahi Aswini K, Carnes Jason, Zíková Alena, Stuart Kenneth D
Seattle Biomedical Research Institute, Seattle, Washington 98109, USA.
RNA. 2009 Feb;15(2):277-86. doi: 10.1261/rna.1353209. Epub 2008 Dec 18.
Mitochondrial (mt) gene expression in Trypanosoma brucei entails multiple types of RNA processing, including polycistronic transcript cleavage, mRNA editing, gRNA oligouridylation, and mRNA polyadenylation, which are catalyzed by various multiprotein complexes. We examined the novel mitochondrial RNA-binding 1 (MRB1) complex that has 16 associated proteins, four of which have motifs suggesting RNA interaction. RNase treatment or the lack of kDNA in mutants resulted in lower MRB1 complex sedimentation in gradients, indicating that MRB1 complex associates with kDNA transcripts. RNAi knockdowns of expression of the Tb10.406.0050 (TbRGGm, RGG motif), Tb927.6.1680 (C2H2 zinc finger), and Tb11.02.5390 (no known motif) MRB1 proteins each inhibited in vitro growth of procyclic form parasites and resulted in cells with abnormal numbers of nuclei. Knockdown of TbRGGm, but not the other two proteins, disrupted the MRB1 complex, indicating that it, but perhaps not the other two, is required for complex assembly and/or stability. The knockdowns resulted in similar but nonidentical patterns of altered in vivo abundances of edited, pre-edited, and preprocessed mt mRNAs, but did not appreciably affect the abundances of mRNAs that do not get edited. These results indicate that MRB1 complex is critical to the processing of mt RNAs, and although its specific function is unknown, it appears essential to parasite viability.
布氏锥虫中的线粒体(mt)基因表达涉及多种类型的RNA加工过程,包括多顺反子转录本切割、mRNA编辑、引导RNA(gRNA)寡聚尿苷酸化和mRNA聚腺苷酸化,这些过程由各种多蛋白复合物催化。我们研究了新型线粒体RNA结合蛋白1(MRB1)复合物,它有16种相关蛋白,其中四种具有提示RNA相互作用的基序。在突变体中进行核糖核酸酶处理或缺乏动基体DNA(kDNA)会导致MRB1复合物在梯度离心中的沉降率降低,这表明MRB1复合物与kDNA转录本相关联。对Tb10.406.0050(TbRGGm,RGG基序)、Tb927.6.1680(C2H2锌指)和Tb11.02.5390(无已知基序)这三种MRB1蛋白的表达进行RNA干扰敲低,每种情况都抑制了前循环型寄生虫的体外生长,并导致细胞核数量异常的细胞出现。敲低TbRGGm,但不是其他两种蛋白,会破坏MRB1复合物,这表明它(而非其他两种蛋白)是复合物组装和/或稳定性所必需的。敲低导致编辑后的、编辑前的和预处理的线粒体mRNA在体内丰度改变的模式相似但不完全相同,但对未编辑的mRNA丰度没有明显影响。这些结果表明,MRB1复合物对线粒体RNA的加工至关重要,尽管其具体功能尚不清楚,但它似乎对寄生虫的生存能力至关重要。