ABSTRACT

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3ʹ-5ʹ exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense (‘mirror’) transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.

摘要

致病酵母白色念珠菌的线粒体基因组显示出由非编码区分隔的几个（八个）初级转录单位的典型组织。编码复合物 I 亚基的基因的存在及其 mtDNA 序列的稳定性使其成为使用转录组学方法研究细胞器基因组表达的有吸引力的模型。负责线粒体中 RNA 降解的主要活性是由DSS1基因编码的酵母中的 3ʹ-5ʹ 外切核糖核酸酶和保守的 Suv3p 解旋酶组成的双组分复合物 (mtEXO)。在白色念珠菌中，缺失DSS1或SUV3基因导致线粒体基因组表达的多种缺陷，导致呼吸能力丧失。转录组学分析揭示了缺乏 mtEXO 活性的突变体中的普遍转录，有证据表明整个基因组被转录，而在野生型菌株中，没有检测到与非编码基因组的重要部分相对应的 RNA。正常线粒体中不存在的反义（“镜像”）转录本在突变体中也很突出。多个成熟转录物的表达，特别是那些从双顺反子 mRNA 翻译的转录物，以及那些含有内含子的转录物在突变体中受到影响，导致蛋白质水平降低和呼吸复合物活性降低。复合体 IV 的表型最为严重，其中成熟 COX1 mRNA 水平降低至约 5% 会导致活性完全丧失。这些结果表明，mtEXO 对 RNA 的降解对于塑造线粒体转录组至关重要，并且是维持转录单位和非编码基因组片段之间的功能分界所必需的。