SUMMARY In Drosophila ovarian germ cells, PIWI-interacting RNAs (piRNAs) direct Aubergine and Argonaute3 to cleave transposon transcripts and instruct Piwi to repress transposon transcription, thereby safeguarding the germline genome. Here, we report that RNA cleavage by Argonaute3 initiates production of most Piwi-bound piRNAs. We find that the cardinal function of Argonaute3, whose piRNA guides predominantly correspond to sense transposon sequences, is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather than to make piRNAs that guide post-transcriptional silencing by Aubergine. We also find that the Tudor domain protein Qin prevents Aubergine’s cleavage products from becoming Piwi-bound piRNAs, ensuring that antisense piRNAs guide Piwi. Although Argonaute3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. This alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
Rhodiola rosea is a commonly used folk medicine for the treatment of high altitude sickness, mountain malhypoxia, and anoxia. Its active ingredient, salidroside [2-(4-hydroxyphenyl)ethyl β-D-glucopyranoside (1)], has been reported to have a broad spectrum of biological effects. However, the protective role of 1 in human erythrocytes remains unclear. This study therefore has investigated the effects of 1 on oxidative stress-induced apoptosis in human erythrocytes (also known as eryptosis or erythroptosis). Compound 1 increased cell survival significantly and prevented human erythrocytes from undergoing eryptosis/erythroptosis mediated by H(2)O(2), as confirmed by the decreased expression of phosphatidylserine on the cell surface and reduced leakage of calcein through the damaged membrane. Mechanistically, 1 was found to exert its protective effects through its antioxidative activity and the inhibition of caspase-3 activation and stress-induced intracellular Ca(2+) rise in a dose-dependent manner. Compound 1 is a protective agent in human erythrocytes against oxidative stress and may be a good adaptogen to enhance the body's resistance to stress and fatigue.
Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster. Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy.
Highlights d The ping-pong and phased piRNA biogenesis pathways are physically separate d Armi couples piRNA biogenesis in nuage to phased piRNA production on mitochondria d Armi binds piRNA precursors during both ping-pong and phased piRNA production d Armi ATPase mutants inappropriately bind mRNA and fail to support piRNA production
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