Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA (‘slicing’), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

中文翻译：

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microRNA Argonautes 的催化残基在秀丽隐杆线虫 microRNA 星链不稳定中发挥适度作用

许多 microRNA (miRNA) 引导的 Argonaute 蛋白可以切割 RNA（“切片”），尽管 miRNA 介导的靶标抑制通常不依赖于切割。在这里，我们使用秀丽隐杆线虫来检查 miRNA Argonautes 的催化残基在生物体发育中的作用。与之前的工作相反，当 CRISPR 引入时，假定的催化残基的突变不会干扰发育。我们发现 miRNA 星链的解旋和衰变在催化残基突变体中存在弱缺陷，在胚胎中观察到的影响最大。Argonaute 样基因 2 (ALG-2) 比 ALG-1 更依赖催化残基进行解旋。对解旋催化残基表现出最大（尽管较小）依赖性的 miRNA 往往会与其星链形成稳定的双链体，并且在某些情况下，降低双链体稳定性会减轻对催化残基的依赖性。虽然一些 miRNA 引导链在突变背景中减少，但其基础尚不清楚，因为变化不依赖于靶向 miRNA 降解 (TDMD) 的效应子 EBAX-1。总的来说，这项工作定义了 miRNA Argonautes 的催化残基在星链衰变中的作用；未来的工作应该检查这种作用是否有助于选择压力，以在后生动物系统发育中保护 miRNA Argonautes 的催化活性。