Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline

Abstract

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.

piRNAs and PIWI proteins: regulators of gene expression in development and stem cells

“…This process is involved in the recognition of expressed (self ) transcripts through transgenerational CSR-1 memory, versus silenced transcripts/foreign sequences (non-self ), in the germline across generations. Recent studies analyzing the rules of piRNA base pairing with mRNAs confirmed the requirement in C. elegans of strong complementarity in the seed region (nucleotides 2-8) followed by an additional match at nucleotides 14-19 (Shen et al, 2018;Zhang et al, 2018).…”

An ancient satellite repeat controls gene expression and embryonic development inAedes aegyptithrough a highly conserved piRNA

“…To assess targeting requirements for tapiR1, we introduced mismatches in the piRNA target sites. Three consecutive mismatches were tolerated unless they were located in the t1 to t9 region of the piRNA (the nucleotides based-paired to piRNA positions 1 to 9) (Fig 2C, Extended Data Fig 6A), and single mismatches only impaired silencing at positions t3 to t7 (Extended data Fig 6A,B), reminiscent of a microRNA seed 35 and comparable to what has been termed the piRNA seed in C. elegans 36,37 . Even though tapiR1 targeting requirements resemble those of miRNAs, the results are unlikely to be due to the piRNA being funnelled into the miRNA pathway.…”

The Caenorhabditis elegans Transgenic Toolbox

“…Because auxin can be washed away, the system is also potentially reversible, although restoration to full protein levels may require a lengthy recovery. Like the methods described above, AID can degrade proteins rapidly and has been used to study the loss-of-function phenotype of several different types of proteins (Zhang et al 2015(Zhang et al , 2018bKerk et al 2017;Pelisch et al 2017;Liu et al 2017a;Patel and Hobert 2017;Yu et al 2017;Shen et al 2018;Ferrandiz et al 2018;Kasimatis et al 2018;Serrano-Saiz et al 2018). One potential issue is that addition of the AID degron tag has been reported in a few cases to interfere with function or stability of the tagged protein even in the absence of TIR1 and auxin (Kerk et al 2017;Patel and Hobert 2017;Schmidt et al 2017).…”