Abstract
BackgroundThe piRNA pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. The Piwi protein-associated small RNAs (piRNAs) guide Piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. Cleavage of the retrotransposon transcript defines at the same time the 5' end of a secondary piRNA that will in turn guide a Piwi protein to a primary piRNA precursor, thereby amplifying primary piRNAs. Although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary piRNAs are not entirely clear.ResultsHere we analyzed small RNAs from three mammalian species, seeking to gain further insight into the mechanisms responsible for the piRNA amplification loop. We found that in all these species piRNA-directed targeting is accompanied by the generation of short sequences that have a very precisely defined length, 19 nucleotides, and a specific spatial relationship with the guide piRNAs.ConclusionsThis suggests that the processing of the 5' product of piRNA-guided cleavage occurs while the piRNA target is engaged by the Piwi protein. Although they are not stabilized through methylation of their 3' ends, the 19-mers are abundant not only in testes lysates but also in immunoprecipitates of Miwi and Mili proteins. They will enable more accurate identification of piRNA loci in deep sequencing data sets.
Highlights
The protein-associated small RNAs (piRNAs) pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing
The 19-mers are not stabilized through methylation of their 3’ termini, and further studies will be required to establish whether or not they have a biological function. It is unclear whether these 19-mers have a biological function or are by-products of piRNA biogenesis. If they were incorporated into Piwi proteins, they would allow the ping-pong mechanism to move on the transcript, instead of being limited to the location defined by the primary piRNA because the cleavage that would be guided by the 19-mer would occur at a different position in the transcript relative to the cleavage that is induced by the secondary piRNA located immediately downstream of the 19-mer
We find a much smaller number of loci with triplex configuration - primary piRNA, secondary piRNA and the 19-mer upstream of the secondary piRNA - in the Mili data (163) compared to the Miwi data (1’846), in spite of the fact that number of perfectly and uniquely mapping sequences that we analyzed was larger for Mili (7’976’050) compared to Miwi (6’517’925)
Summary
The piRNA pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. The Piwi protein-associated small RNAs (piRNAs) guide Piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. Some of the Piwi-associated small RNAs (piRNAs) are involved in regulating the expression of transposable elements, a function which is important during genome activation in the germ line. Evidence for this specific role of piRNAs comes from the studies in y [5,6,7], zebrafish [8,9] and mouse [10,11,12,13].
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