Ribogenetics@Biochemistry Lab, School of Engineering and Science, MOLIFE Research Center, Jacobs University Bremen, Bremen, Germany.
PLoS One. 2013 May 20;8(5):e64804. doi: 10.1371/journal.pone.0064804. Print 2013.
RNA interference (RNAi) is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs) are required for RNAi. To study the function of the three RdRPs in the amoeba Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-gal small interfering RNAs (siRNAs) could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5' of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.
RNA 干扰 (RNAi) 是真核生物中的一种基因调控机制，基于双链 RNA 的存在，可作用于转录或转录后水平。在许多物种中，RNA 依赖性 RNA 聚合酶 (RdRP) 是 RNAi 所必需的。为了研究三种 RdRP 在变形虫 Dictyostelium discoideum 中的功能，我们已在所有可能的组合中删除了编码基因 rrpA、rrpB 和 rrpC。在这些菌株中，针对转基因 lacZ 的反义或发夹 RNA 构建体的表达导致 β-半乳糖苷酶活性降低 50%。令人惊讶的是，Northern 印迹显示 lacZ mRNA 水平不变，表明存在转录后调控。只有在 rrpC 敲除株中，在表达反义 RNA 的菌株中才能检测到低水平的 β-半乳糖小干扰 RNA (siRNA)。与此相反，并且在更高的水平上，所有表达发夹 RNA 的菌株都具有 β-半乳糖 siRNA。在所有菌株中观察到沉默信号向原始发夹触发的 5'端的 mRNA 序列传播，除了 rrpC 基因或与 dicer 相关的核酸酶 DrnB 缺失的情况。因此，我们的数据表明，D. discoideum 中存在 RNA 沉默信号的转导性，并且它需要两种酶 RrpC 和 DrnB。
