Hierarchical rules for Argonaute loading in Drosophila. Drosophila melanogaster

Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway. Overall design: These were independently processed and sequenced using the Illumina GAII platform. In total, five libraries were analyzed.

果蝇Argonaute蛋白（Argonaute）家族的AGO1与AGO2在功能及结合的小RNA组分上存在显著差异。AGO2偏好结合小干扰RNA（small interfering RNA，siRNA），而AGO1则几乎仅特异性结合微RNA（microRNA，miRNA）。微RNA双链具有内在的结构不对称性：其中一条链（即miR链）优先被装载进入AGO1，通过碱基互补识别靶信使RNA（messenger RNA，mRNA）并调控其表达；另一条链（miR*）此前被认为仅是微RNA生成过程中的副产物。本研究证实，miR*常以具有功能活性的分子形式被装载进入AGO2。这一发现表明，每一个微RNA前体均可潜在产生两条成熟小RNA链，并在RNA干扰（RNA interference，RNAi）通路中被差异化分选。miR*的生成依赖于经典的微RNA生成通路，但其被装载进入AGO2的过程，则由传统上负责小干扰RNA装载的宿主因子所介导。通过推导并验证可预测AGO蛋白差异化装载的层级规则，我们发现加工后的双链RNA的结构与热力学特性等内在决定因素，可调控每条RNA链在RNAi通路中的最终命运。总体实验设计：所有样本均采用Illumina GAII测序平台进行独立处理与测序，共分析了5个测序文库。