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RNA: methods and protocols - a new series

This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze nucleic acids and proteins

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour

A 5′-uridine amplifies miRNA/miRNA* asymmetry in Drosophila by promoting RNA-induced silencing complex formation

International audienceBACKGROUND: MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U). RESULTS: Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5'-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5'-U promotes miRNA loading while decreasing the loading of the miRNA*. CONCLUSION: We suggest that recognition of the 5'-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5'-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5'-nt

PIWI-interacting RNAs:small RNAs with big functions

In animals, PIWI-interacting RNAs (piRNAs) of 21–35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.</p