Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum. Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts accumulate in a drnB knock-out strain, generally preceded by a T-rich putative promoter motif. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. Different biogenesis pathways generate the 3’ ends of these pri-miRNAs, where pri-mir-1177 reads into the downstream gene, while pri-mir-1176 has a distinct non-polyadenylated end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development. Overall design: small and poly(A) enriched RNA-seq of D. discoideum AX2 WT and drnB- knock out strain from vegetative growing and 16h developed cells.

微小核糖核酸（miRNA）可在诸多真核生物中调控基因表达，参与控制多种生物学过程。其从初级转录本到成熟miRNA的生物发生过程，已在动物与植物中得到广泛解析，且这两类系统发育相距甚远的生物类群间存在显著差异。然而，对于进化位置介于动植物之间的生物，以及单细胞生物中的miRNA生物发生过程，目前仍知之甚少。本研究针对单细胞变形虫盘基网柄菌（*Dictyostelium discoideum*，隶属于变形虫门Amoebozoa）的miRNA成熟过程展开探究，该类群的演化分支时间晚于植物但早于动物。通过对小RNA及聚腺苷酸（poly(A)）筛选RNA进行高通量测序，研究证实类Dicer蛋白DrnB是盘基网柄菌全局miRNA成熟过程所必需的核心蛋白，且基本具有特异性。本研究的RNA测序（RNA-seq）数据还显示，在drnB敲除菌株中，更长的miRNA转录本发生积累，这类转录本的上游通常存在富含胸腺嘧啶（T）的推定启动子基序。针对两种模式miRNA，本研究明确了其初级（pri-miRNA）的转录起始位点（TSS），并证实这类pri-miRNA携带RNA聚合酶II特异性的m7G帽子结构。不同的生物发生途径可生成这些pri-miRNA的3’末端：pri-mir-1177的转录会通读至下游基因，而pri-mir-1176则具有独特的非聚腺苷酸化末端。该3’末端会被加工为较短的中间产物，这类中间产物在DrnB缺失的细胞中更为稳定，其中部分带有短的寡聚腺苷酸（oligo(A)）尾。此外，本研究还鉴定出10种全新的miRNA，这些miRNA均依赖DrnB且受发育过程调控。由此可见，盘基网柄菌的miRNA调控系统兼具动植物的特征，这与其演化位置相符，同时也可能是对其复杂生活史——单细胞生长与多细胞发育——的一种适应。实验整体设计：对盘基网柄菌AX2野生型（WT）与drnB敲除菌株，分别在营养生长期与发育16小时的细胞中开展小RNA及聚腺苷酸富集RNA的高通量测序。