XenopusPiwi proteins interact with a broad proportion of the oocyte transcriptome

Abstract

Piwi proteins utilize small RNAs (piRNAs) to recognize target transcripts such as transposable elements (TE). However, extensive piRNA sequence diversity also suggests that Piwi/piRNA complexes interact with many transcripts beyond TEs. To determine Piwi target RNAs, we used ribonucleoprotein-immunoprecipitation (RIP) and cross-linking and immunoprecipitation (CLIP) to identify thousands of transcripts associated with the Piwi proteins XIWI and XILI (Piwi-protein-associated transcripts, PATs) from early stage oocytes of and Most PATs associate with both XIWI and XILI and include transcripts of developmentally important proteins in oogenesis and embryogenesis. Only a minor fraction of PATs in both frog species displayed near perfect matches to piRNAs. Since predicting imperfect pairing between all piRNAs and target RNAs remains intractable, we instead determined that PAT read counts correlate well with the lengths and expression levels of transcripts, features that have also been observed for oocyte mRNAs associated with Piwi proteins. We used an in vitro assay with exogenous RNA to confirm that XIWI associates with RNAs in a length- and concentration-dependent manner. In this assay, noncoding transcripts with many perfectly matched antisense piRNAs were unstable, whereas coding transcripts with matching piRNAs were stable, consistent with emerging evidence that Piwi proteins both promote the turnover of TEs and other RNAs, and may also regulate mRNA localization and translation. Our study suggests that Piwi proteins play multiple roles in germ cells and establishes a tractable vertebrate system to study the role of Piwi proteins in transcript regulation.

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

“…The presence of a large repertoire of TE-independent piRNAs in adult testes is conserved in many species, including hen, rat, pig, marmoset and human (Gebert et al, 2015;Hirano et al, 2014;Lau et al, 2006;Li et al, 2013;Williams et al, 2015). In addition, PIWI proteins bind a large proportion of endogenous mRNAs in oocytes from two Xenopus species (Toombs et al, 2017). This set of data indicates a conserved biological function of piRNAs in posttranscriptional regulation of developmental programs.…”

The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes

“…Crosslinking immunoprecipitation (CLIP) analyses of PIWIs suggest that they associate with diverse mRNAs (8–10). However, because diverse piRNAs engage with many mRNAs, it is difficult to infer the target of a given piRNA from these CLIP analyses.…”

“…However, these cleaved mRNAs likely present only a fraction of piRNA targets in vivo , since the slicer activity of PIWI is dispensable for silencing in some animals, including C. elegans (13–16). As only few piRNA targets other than transposons have been identified, the piRNA targeting rules remain undefined and both sequence-specific and sequence-nonspecific functions of PIWI/piRNA complex have been proposed (8–10, 12, 17, 18). …”

Small Noncoding RNA Expression in Cancer

“…In addition, many piRNAs do not match transposon sequences, suggesting an even greater set of targets and functional roles for piRNAs [80]. In fact, cross-linking immunoprecipitation (CLIP) analyses unveils many nontransposon mRNAs engaged with PIWI proteins [81]. In Caenorhabditis elegans, it was previous demonstrated that piRNA action is analogous to miRNAs, in that seed sequences are required for mRNA targeting, but unlike miRNAs, piRNAs do not tolerate many mismatches out of this region [82].…”