Structural insights into piRNA recognition by the human PIWI‐like 1 PAZ domain

Major advancements have been made in the past decade towards understanding the regulatory role of small non-coding RNAs (ncRNAs) in diverse development processes1. An integral part of this regulation is the conserved PIWI/Argonaute protein family. Phylogenetic analysis indicates that this protein family can be divided into two sub-families: the Piwi sub-family, named after Drosophila melanogaster protein PIWI (P-element induced wimpy testis), and the Ago sub-family, based on its founding member in Arabidopsis thaliana (Ago). Ago proteins are involved in RNA-mediated gene silencing complexes and utilize short guide RNAs to target specific nucleic acids1-4, whereas Piwi proteins bind to a novel class of ncRNAs called PIWI-interacting RNAs (piRNAs) that play diverse functions in germline development and gametogenesis5-11. Piwi/Ago proteins have a molecular weight of about 90 kDa and are defined by three protein motifs: the PAZ, MID and PIWI domains. The N-terminal PAZ domain is accountable for binding the 3′-end of the guide strand RNA; the middle MID domain is responsible for binding the 5′-phosphate RNA; and the following C-terminal PIWI domain has an RNase H endonuclease fold that is responsible for the RNA cleavage. The best-characterized small RNA partners of Ago proteins are microRNAs (miRNAs). Ago proteins utilize miRNAs to silence genes post-transcriptionally or use small-interfering RNAs (siRNAs) in both transcription and post-transcription silencing mechanisms12-17. However, Piwi proteins are restricted to germline cells and stem cells, and have been found to interact with a novel class of piRNAs (∼28-33 nucleotides) longer than miRNAs and siRNAs (∼20 nucleotides), suggesting novel functions distinct from those of Ago proteins18-22. piRNAs are 28 to 33 nucleotides in length and have been characterized by the cloning of small RNAs from anti-Piwi immunoprecipitates prepared from mammalian testes7-9. As the proportion of repeat elements able to generate piRNAs is actually smaller within the piRNA regions than the frequency of repeat sequences in the mouse genome, they are not believed to be derived from repeat sequences. It is believed that piRNAs are processed from single-stranded primary transcripts that are transcribed from defined genomic regions and have a preference for a uridine at their 5′ terminal6-10,23. Mammalian piRNAs are a highly complex mix of sequences, with tens of thousands of distinct piRNAs generated from the 50 to 100 defined primary transcripts. This may suggest that mammalian piRNAs, unlike miRNAs, are not processed in a precise manner. However, approximately 20% of all piRNA sequences were cloned three or more times, and many piRNA sequences from the same strand are partially overlapping, suggesting a quasi-random mechanism. The mechanism of biogenesis of D. melanogaster rasiRNAs is beginning to be elucidated, and may offer parallels for a specific mode of processing for piRNAs as well. piRNA biogenesis is thought to be Dicer-independent and they appear to be 2′-O'methylated at their 3′ terminal24-26. Between mammals, mature piRNAs are not conserved; however, the genomic regions, from which they derive, in particular the promoter sequences, are conserved. Mammalian piRNAs are strongly expressed in the male germline, their total number per cell obtained from testis tissue reaching up to two million – about 10-fold higher than the miRNA content of these cells. Despite their importance in developmental biology, a mechanistic understanding of the biogenesis of piRNAs is lacking, and the specific functional role PIWI proteins play in piRNA generation remains elusive. To understand the molecular basis of piRNA recognition by PIWI proteins, we have determined the three-dimensional solution structures of the human PIWI-like 1 (hPIWIL1) PAZ domain in its free form, as well as bound to a ssRNA (5′-pUGACA) oligonucleotide using nuclear magnetic resonance (NMR) spectroscopy methods.