In fruit flies, a few very large genes generate the small RNAs that silence parasitic DNA elements. These RNAs might also participate in an amplification circuit that increases their potency.
Nearly half of the human genome and a third of the fruit fly's consists of selfish elements called transposons, 'jumping genes' that insert themselves into new locations, mutating other genes and damaging chromosomes. These molecular parasites include simple nucleotide repeats and virus-like elements that have colonized genomes throughout evolution. In response, animals have evolved complex mechanisms to silence transposons. The fruit fly Drosophila melanogaster silences its transposons by a mechanism that uses small RNA 'guides'. Reporting in Cell1 and Science2, two research groups propose an unanticipated explanation for the production and amplification of these small, silencing RNAs.
The best known silencing pathway guided by small RNAs is RNA interference (RNAi), in which small interfering RNAs (siRNAs) trigger an immune response that defends plants and animals against viruses by destroying complementary RNA sequences3,4. The siRNAs bind to members of the Argonaute family of proteins — molecular 'scissors' that use the small RNA guide to bind to and cut a second RNA molecule, the 'target'. The use of RNA guides rather than protein antibodies is a key difference between the sequence-based RNAi defence pathway and our adaptive immune responses, which recognize viral proteins. The enzyme Dicer produces siRNAs by cutting long, double-stranded RNA into smaller pieces about 21 nucleotides long. One strand of each siRNA is then loaded onto an Argonaute protein, generating a RNA–protein complex that binds to a viral RNA target by base-pairing and cuts it.
Like viruses, transposons are parasites, but, unlike viruses, they are primarily transmitted by inheritance, rather than through infection. To produce enzymes that facilitate their jumping to a new location in the genome, transposons must first be copied into mRNA. The evolution of small-RNA-guided silencing mechanisms, which prevent copying of transposons into RNA sequences, allows higher organisms to defend their genes against transposons.
In some higher organisms, the RNAi pathway provides a crucial defence mechanism against transposons5,6,7. But for fruit flies the genome is guarded by the Piwi-associated interfering RNA (piRNA) pathway8. At the heart of this pathway lies a specialized set of Argonaute proteins produced in the germ cells. Flies have five Argonaute proteins: Ago1, which uses small RNAs called microRNAs to regulate gene expression; Ago2, which uses siRNAs to fight viral infection; and three closely related Piwi proteins — Piwi, Aubergine and Argonaute3 (Ago3) — which use piRNAs to silence transposons and other parasitic DNAs. In flies, piRNAs are also called repeat-associated siRNAs, or rasiRNAs.
The Hannon1 and Siomi2 laboratories separately set out to identify small RNAs bound to each of the three fly Piwi proteins, in the hope that their sequences would reveal how rasiRNAs are made and function. Their findings are simply spectacular, suggesting that rasiRNAs arise from a small number of trigger loci — huge 'genes' that produce small RNAs against many selfish genetic elements — and that they are amplified through reciprocal cycles of cleavage by pairs of Piwi proteins (Fig. 1, overleaf).
To identify the RNAs bound to the three Drosophila Piwi proteins, each laboratory used antibodies to purify Piwi, Aubergine and Ago3 from the fly's ovaries, along with their associated rasiRNAs. Hannon and colleagues1 identified 60,691 different rasiRNAs. It is difficult to pinpoint the site of origin of any one rasiRNA to a single genomic location, because nearly identical copies of specific transposons litter the entire genome. However, the set of rasiRNA sequences identified by these authors was so large that around 12,000 could be assigned to unique sites. These sites formed just 142 clusters, with a single cluster on the right arm of chromosome 2 comprising about 21% of all the unique rasiRNAs.
Another cluster corresponded to a genetic locus called flamenco on the X chromosome, which was previously shown to repress the jumping of the gypsy, Idefix and ZAM transposons. Since its discovery9, flamenco has posed a puzzle because no protein-encoding gene resides at this locus. The new results indicate that flamenco, instead of producing a protein, is the source of rasiRNAs that target multiple types of transposon. These rasiRNAs may come from an enormous precursor RNA molecule, as mutations lying at the beginning of the locus disrupt flamenco-derived rasiRNAs some 168,000 nucleotides away. Loss of flamenco function activates transposons, such as gypsy, that lie within it, although most gypsy transposons reside outside this locus. However, it has no effect on transposons that are not found in flamenco. Thus, flamenco seems to have evolved as a master regulator of gypsy, Idefix and ZAM transposons and is the primary, if not the sole, source of rasiRNAs against these selfish elements.
Most rasiRNAs correspond to the antisense strand of transposons and can therefore bind to, and presumably destroy, the RNA transcripts of transposons1,8. These antisense rasiRNAs bind to Piwi and Aubergine. The two new papers1,2 report that the small RNAs bound to Ago3 are nearly all of the sense orientation. Of the 353 Ago3-associated rasiRNAs identified by Siomi's group2, the first 10 nucleotides of 16 sequences could be paired with rasiRNAs bound to Aubergine. And more than 11,200 (48%) of the Ago3-associated sense rasiRNAs identified by Hannon's group1 could form an offset couple with at least one antisense rasiRNA.
A 10-nucleotide offset between the beginning of a small RNA guide and a second RNA molecule has a special meaning. Argonaute proteins cut target RNAs by measuring 10 nucleotides from the beginning of their RNA guide to the site of cleavage on their target. Such a pairing scheme suggests that the starting nucleotide of each antisense rasiRNA is defined by a cut that is guided by a corresponding sense rasiRNA. Reinforcing this view, nearly all the antisense rasiRNAs begin with the nucleotide U, whereas the sense rasiRNAs show no bias for beginning with U, A, C or G. Instead, the tenth nucleotide of the sense rasiRNAs was almost always A, which would allow it to pair with the first nucleotide — U — of an Aubergine- or Piwi-bound antisense rasiRNA (Fig. 1b).
Imagine, then, that a mother fly protects her offspring by providing her developing eggs with some of her Aubergine- and Piwi-bound antisense rasiRNAs. These could then generate sense rasiRNAs by cleaving the RNA transcripts of transposons, thereby simultaneously silencing them and initiating a cycle of rasiRNA amplification. The sense rasiRNAs bound to Ago3 would then cleave the long, antisense transcripts produced by master regulatory loci such as flamenco, producing new antisense rasiRNAs that would bind to Aubergine or Piwi. If antisense transcripts from master regulatory loci are generally more abundant than sense transcripts from transposons — a reasonable assumption as transposons are normally silenced — then the pool of rasiRNAs would, as observed, be disproportionately antisense.
Of course, the model proposed by these authors1,2 only explains how the start of each rasiRNA is defined. How the 3′ end of the rasiRNA is made remains to be discovered. And what of piRNAs in humans? piRNAs were recently discovered in immature mouse, rat and human sperm cells10, and in zebrafish testes and ovaries11, although they were mostly not associated with transposon sequences. What piRNAs do in mammalian sperm is unknown, but, like fly piRNAs, they derive from large genomic clusters. And like fly piRNAs8, they do not seem to be made by Dicer. Perhaps all piRNAs are made and amplified by reciprocal cycles of Piwi-catalysed slicing of sense and antisense transcripts. Stay tuned for further detailed sequence analyses.
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Enhancement of siRNA transfection by the optimization of fatty acid length and histidine content in the CPP
Biomaterials Science (2019)
Molecular Biology and Evolution (2016)
Molecular Immunology (2015)
Human Molecular Genetics (2014)
National Science Review (2014)