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Xenopus Piwi Proteins Interact with a Broad Proportion of the Oocyte Transcriptome

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Xenopus Piwi Proteins Interact with a Broad Proportion of the Oocyte Transcriptome Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Xenopus Piwi proteins interact with a broad proportion of the oocyte transcriptome JAMES A. TOOMBS,1,2,4 YULIYA A. SYTNIKOVA,3,5 GUNG-WEI CHIRN,3,6 IGNATIUS ANG,3,7 NELSON C. LAU,3 and MICHAEL D. BLOWER1,2 1Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA 2Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA 3Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02454, USA 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 X. laevis and X. tropicalis. 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 Drosophila 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. Keywords: Piwi; Xenopus; oocyte; XIWI; XILI INTRODUCTION (Juliano et al. 2011; Siomi et al. 2011; Clark and Lau 2014). The base-pairing interactions between piRNAs and TE tran- Animal oocytes are generally large cells containing large scripts allows Piwi proteins to directly repress TEs, thus quantities of maternally deposited mRNAs and proteins preserving the integrity of the germ cell genome (Han et al. that are required to drive several rounds of cell division prior 2015; Mohn et al. 2015; Senti et al. 2015; Wang et al. 2015; to the onset of zygotic transcription (Tadros and Lipshitz Yang et al. 2016). 2009). Among the most important maternally deposited However, a second, less understood function for Piwi pro- ribonucleoproteins (RNPs) are germ cell enriched RNPs be- teins is to serve as a regulator of RNAs in the germ plasm longing to the RNA interference (RNAi) pathway, such as the (Harris and Macdonald 2001; Findley et al. 2003; Megosh Piwi pathway. Piwi proteins are conserved from basal animals et al. 2006), an asymmetrically localized compartment in to vertebrates (Grimson et al. 2008) and bind to short RNAs animal oocytes composed of maternal RNAs and proteins called Piwi-interacting RNAs (piRNAs), many of which have that is later segregated into germ cell blastomeres (Strome complementarity to transposable element (TE) sequences and Updike 2015). Many important maternal RNAs in germ plasm are tightly regulated during oogenesis and em- 4Present address: Brigham Research Institute, Brigham and Women’s bryogenesis to ensure proper development of the zygote. Hospital, Boston, MA 02115, USA 5 Although Piwi proteins and piRNAs are also enriched in Present address: Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA 6Present address: iBioinfo Group LLC, Lexington, MA 02421, USA 7Present address: University of New England School of Osteopathic © 2017 Toombs et al. This article is distributed exclusively by the RNA Medicine, Biddeford, ME 04005, USA Society for the first 12 months after the full-issue publication date (see Corresponding authors: [email protected], [email protected]. http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is harvard.edu available under a Creative Commons License (Attribution-NonCommercial Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.058859. 4.0 International), as described at http://creativecommons.org/licenses/by- 116. nc/4.0/. 504 RNA 23:504–520; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Xenopus Piwi-protein-associated transcripts animal germ plasms, it remains poorly understood if Piwi/ mouse can associate broadly with protein-coding mRNAs piRNA complexes are involved in the regulation of these ma- as well as long-noncoding RNAs, including in a piRNA-inde- ternal RNAs, because many mutations in Piwi-pathway genes pendent fashion (Vourekas et al. 2012, 2016; Sytnikova et al. severely compromise early steps of gonadogenesis (Lin and 2014; Barckmann et al. 2015). This broad association of Spradling 1997; Cox et al. 1998; Harris and Macdonald mRNAs with Piwi proteins was most recently observed in 2001; Findley et al. 2003), and thus hinder the study of later Drosophila oocyte germ plasm (Barckmann et al. 2015; stage gametes. Vourekas et al. 2016), with a newly emerging hypothesis pro- Although many piRNAs contain TE sequences, an equally posing that piRNA base-pairing requirements are so loose large if not greater number lack sequence homology with TEs that transcript association with Piwi proteins may depend because there are millions of unique piRNAs that are gener- upon its length and its abundance (Vourekas et al. 2016). ated from many genomic loci that can be depleted of TE We previously showed that several oocyte mRNAs associ- sequences (Girard et al. 2006; Lau et al. 2006; Li et al. 2013; ate with the X. tropicalis protein, XIWI (also known as Chirn et al. 2015). With this tremendous piRNA sequence PIWIL1 by the HUGO nomenclature) (Lau et al. 2009a). diversity, the guiding mechanism of Piwi/piRNA complexes To comprehensively identify Xenopus oocyte PATs, we first is unclear if imperfect base-pairing is considered (Post et al. characterized the expression and oocyte localization of the 2014). Additionally, the intrinsic RNA-binding activities of XIWI ortholog, XILI (also known as PIWIL2). We then uti- Piwi proteins could further broaden the range of Piwi-pro- lized XIWI and XILI antibodies to perform RIP-seq and tein associated transcripts (PATs) (Sytnikova et al. 2014). CLIP-seq in X. laevis and X. tropicalis oocytes, respectively, Therefore, understanding the regulatory mechanisms of ani- to generate extensive lists of Xenopus PATs and determine mal Piwi proteins during gametogenesis requires a complete how many transcripts interact with both XIWI and XILI be- catalog of PATs across multiple animal species. tween these two frog species. Additionally, we characterized RNP-immunoprecipitation (RIP) and cross-linking im- X. laevis oocyte piRNA populations and define intergenic munoprecipitation (CLIP) are common methods that have piRNA cluster loci within the tetraploid genome of X. laevis. provided tremendous insight into transcripts associated with Finally, we demonstrate the capacity of an X. laevis oocyte various proteins and preferred binding sites of RNA-binding extract to test for the stability of particular PATs with abun- proteins (Ule et al. 2003; Keene et al. 2006; Licatalosi et al. dant amounts of mapping piRNAs as well as the capacity of 2008). A common goal of RNA target profiling experiments exogenous RNA to interact with XIWI. This study expands on Argonaute (AGO) and Piwi proteins (Chi et al. 2009; our knowledge about the role of Piwi protein in Xenopus Hafner et al. 2010; Zisoulis et al. 2010; Leung et al. 2011; species, and provides a platform for future dissection of the Vourekas et al. 2012, 2016; Sytnikova et al. 2014; Barckmann Piwi-protein-dependent regulatory mechanisms for mater- et al. 2015) has been to develop the set of sequence-recogni- nally deposited RNAs in Xenopus oocytes. tion preferences between these RNP, the small RNAs bound within these RNPs, and their associated target mRNA tran- RESULTS scripts. The AGO protein studies have reinforced mecha- nisms of miRNAs targeting 3′ untranslated regions (3′ ′ Characterization of X. laevis Piwi proteins, XIWI UTRs) in mRNAs by the 5 end “seed sequence” of the and XILI miRNAs, and revealed additional motifs in mRNAs that AGO proteins can bind independently of the miRNA (Chi Having previously characterized XIWI expression and local- et al. 2009, 2012; Hafner et al. 2010; Zisoulis et al. 2010; ization in X. tropicalis (Lau et al. 2009a), we then turned our Leung et al. 2011; Baigude et al. 2012; Clark et al. 2014; attention to its homolog, XILI, which is well expressed in X. Grosswendt et al. 2014). The immense diversity of piRNA laevis oocyte mRNA and protein profiles (Wilczynska et al. sequences across animals continues to present a bioinfor- 2009; Wuhr et al. 2014). Using new gene models for X. laevis
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