Genetic and mechanistic diversity of piRNA 3′-end formation

Abstract

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation.

piRNA Biogenesis in Drosophila melanogaster

“…3’ end formation can also be induced by slicer (ping-pong) cleavage. In this case, the mature piRNA is generated by two closely spaced slicer cleavages [77]. Finally, 3’ end formation might require processing by exonuclease(s).…”

“…In this case, a 3’-to-5’ exonuclease trims a longer precursor formed by cleavage by an endonuclease (slicer or Zuc) to make mature piRNAs of the correct size. Such ‘Trimmer’ activity was first observed in vitro using lysate from a silkworm cell line [78] and later associated with the exonuclease Nibbler (Nbr) [77, 79, 80]. Independently of the mechanism by which the 3’end of piRNAs is generated, the last step is the 2’OMe-modification of the last nucleotide by Hen1, which is thought to increase the stability of piRNAs [80–82].…”

“…The model relies on the observation that a transcript that is recognized by complementary piRNAs residing in Aub or Ago3 is first cleaved by their slicer activities to generate a single responder piRNA followed by Zuc-dependent processing of the remainder of the transcript to multiple piRNAs [72, 73]. Insertion of a single piRNA target sequence into a heterologous transcript leads to efficient processing of the transcript into piRNAs [73, 77, 108]. This model raises the obvious question of how the very first piRNAs – which subsequently recognize piRNA precursors - are made.…”

The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

“…Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions, and 2S rRNA was depleted as described (Hayashi et al, 2016). Small RNA libraries were generated as described (Jayaprakash et al, 2011).…”

Small Non-Coding RNAs in Regulation of Course and Therapeutic Efficacy in Acute Myeloid Leukemia

“…Recently, it was shown that high expression of piwi-like protein 4 in leukemic cells in 72% of the patients with different types of AML compared to healthy controls, and in 90% of the patients with MLL-AF9 rearranged AML [13,65]. PiRNAs act as sequence-specific guides for piwi proteins, and they support biogenesis and stability of piRNAs.…”