Université Paris-Sud, Orsay, France.
PLoS One. 2011 Jan 25;6(1):e16325. doi: 10.1371/journal.pone.0016325.
In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a N(in)-C(out) topology. Plasmid-versions of 5' end or 3' end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5' flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5' and 3' ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats.
在真核生物中，核核糖体 DNA（rDNA）是结构 18S、5.8S 和 25S rRNA 的来源。在半子囊菌酵母中，描述了 25S rDNA 序列容纳了一个反义开放阅读框（ORF），命名为 TAR1，代表核糖体 RNA 的转录反义。在这里，我们首次对这种非典型酵母基因的真实产物进行了免疫检测和亚细胞定位。使用针对酿酒酵母 TAR1 产物预测的氨基酸序列的特异性抗体，我们在酿酒酵母（Sc）和乳酸克鲁维酵母（Kl）物种中检测到了内源性 Tar1p 多肽，并发现这两种蛋白质都定位于线粒体。对纯化的线粒体进行蛋白酶和碳酸盐处理进一步表明，内源性 Sc Tar1p 蛋白在内膜中以 N（in）-C（out）拓扑结构亚定位。使用 5'端或 3'端截断 TAR1 ORF 的质粒版本证明，Sc Tar1p 的 N 端和 C 端都不是其定位所必需的。此外，Tar1p 是一种无前导肽的蛋白质。发现内源性 Sc Tar1p 是一种低丰度的蛋白质，在可发酵和不可发酵的生长条件下表达。还发现内源性 Sc TAR1 转录物丰度也较低，并且当在荧光素酶报告系统中进行测定时，TAR1 ORF 的 5'侧翼区表现出适度的启动子活性。使用快速扩增 cDNA 末端（RACE）PCR，我们还确定内源性 Sc TAR1 转录物具有异质的 5'和 3'末端，可能反映了嵌入在活跃转录的 rDNA 序列中的基因的复杂表达。总之，我们的结果明确确定，反义酵母基因 TAR1 在核 rDNA 重复序列中构成了一个功能性转录单位。
