Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis

Fernandes, Maria Gomes; He, Nannan; Wang, Fang; Iperen, Liesbeth van; Eguizábal, Cristina; Matorras, Roberto; Lopes, Susana M. Chuva de Sousa

doi:10.1093/humrep/dex365

Cited by 44 publications

(52 citation statements)

References 49 publications

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“…Our study provides the most comprehensive characterization of the PIWI-piRNA pathway in the human fetal testis to date. In addition to confirming and expanding upon the reported expression of HILI and HIWI2 (Gomes Fernandes et al 2018), we observed dynamic cytoplasmic to nucler localization of HIWI2 between GW18 -21 (Figure 1). Recent studies in human fetal ovary and adult testis showed that piRNAs were derived from genomic piRNA clusters (Ha et al 2014;Roovers et al 2015;Williams et al 2015).…”

Section: Discussionsupporting

confidence: 87%

“…Initially low levels of HILI at GW 11 increased through development, and protein was distributed throughout the cytoplasm as well as in puncta beginning at GW13, consistent with reported localization of MILI to pi-bodies, or intermitochondrial cement, in mice ). HIWI (PIWIL1) expression was not observed in fetal testes (Figure S1B), as expected based on its known adult-specific expression in human (Gomes Fernandes et al 2018) and mouse (Deng and Lin 2002) (Figure S1C). HIWI2…”

Section: Dynamic Expression and Subcellular Localization Of Piwi Homosupporting

confidence: 81%

“…Notably, piRNAs identified are derived from clusters and resemble the composition of mouse pachytene piRNAs rather than fetal pre-pachytene piRNAs which are largely transposon-derived (Aravin et al Page 3 2007;Kuramochi-Miyagawa et al 2008). In human fetal testis, piRNA expression has been difficult to detect due to low abundance (Williams et al 2015;Gainetdinov et al 2017), and reported localization of PIWIL2 (HILI) and PIWIL4 (HIWI2) in the cytoplasm does not support nuclear silencing by this pathway (Gomes Fernandes et al 2018).…”

Section: Introductionmentioning

confidence: 99%

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Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells

Reznik

Cincotta

Jaszczak

et al. 2018

Preprint

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Epigenetic resetting in germ cells during development leads to the de-repression of transposable elements (TEs). piRNAs protect fetal germ cells from potentially harmful TEs by targeted destruction of mRNA and deposition of repressive epigenetic marks. Here we provide the first evidence for an active piRNA pathway and TE repression in germ cells of human fetal testis. We identify pre-pachytene piRNAs with features of secondary amplification that map most abundantly to L1 family TEs. We find that L1-ORF1p expression is heterogeneous in fetal germ cells, peaks at mid-gestation and declines concomitantly with increasing levels of piRNAs and H3K9me3, as well as nuclear localization of HIWI2. Surprisingly, following this decline, the same cells with accumulation of L1-ORF1p display highest levels of HIWI2 and H3K9me3, whereas L1-ORF1p low cells are also low in HIWI2 and H3K9me3. Conversely, earlier in development, the germ cells lacking L1-ORF1p express high levels of the chaperone HSP90a.We propose that a subset of HSP90a-armed germ cells resists L1 expression, whereas only those vulnerable L1-expressing germ cells activate the PIWI-piRNA repression pathway which leads to epigenetic silencing of L1 via H3K9me3.

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Section: Discussionsupporting

confidence: 87%

Section: Dynamic Expression and Subcellular Localization Of Piwi Homosupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

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Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells

Reznik

Cincotta

Jaszczak

et al. 2018

Preprint

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“…Since the subcellular localization of a protein often dictates the mode of action and accessibility to substrates and cofactors, we sought to trace the intracellular distribution of PIWIL3 over developmental stages, to further elucidate the functional role of PIWIL3. While the intracellular distributions of PIWIL1, PIWIL2 and PIWIL4 have been elucidated in mouse testis 10,15 and human fetal oocytes 14 , the subcellular compartment(s) in which PIWIL3 exerts its function remains uninvestigated to date. Herein, we follow the distribution and expression pattern of PIWIL3 in bovine oocytes and preimplantation embryos, by transiently expressing EGFP-PIWIL3 fusion proteins.…”

Section: Resultsmentioning

confidence: 99%

“…This appears to imply that products of Piwi genes (homologues of human PIWIL1, PIWIL2, PIWIL4 ) are not critically needed during oogenesis, maturation and post-fertilisation, at least in the murine system. We have previously established that human, macaque and bovine oocytes express large amounts of piRNAs, and 30-50% of these are enriched for transposon sequences, in particular, the expression of a fourth PIWI gene, PIWIL3 , was detected in human and bovine species but absent in murine systems 13,14 . Due, in part, to the perennial shortage of PIWIL3-specific antibodies, and the lack of mammalian species expressing PIWIL3 that are amenable to genetic manipulations, little is known about the function and subcellular localization of PIWIL3 as well as biogenesis in PIWIL3 related piRNAs.…”

Section: Introductionmentioning

confidence: 99%

PIWIL3 forms a complex with TDRKH in mammalian oocytes

Tan

Tol

Rosenkranz

et al. 2019

Preprint

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18PIWIs are crucial guardians of genome integrity, particularly in germ cells. While mammalian 19 PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene 20 is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot 21 be effectively modeled by mouse knockouts. Herein, we investigated the expression, 22 distribution and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, 23 and demonstrated that PIWIL3 expression is stringently controlled both spatially and 24 temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-25 recruited three-membered complex with TDRKH and PNLDC1, and demonstrated by 26 mutagenesis that PIWIL3 N-terminal arginine modifications are required for complex 27 assembly. Finally we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here 28 that about 50% of these piRNAs map to transposable elements, recapitulating the important 29 role of PIWIL3 in maintaining genome integrity in mammalian oocytes. 30 65 5 Results 66 PIWIL3 is expressed in the cytoplasm during oocyte maturation and early embryo 67 development 68 Since the subcellular localization of a protein often dictates the mode of action and 69 accessibility to substrates and cofactors, we sought to trace the intracellular distribution of 70 PIWIL3 over developmental stages, to further elucidate the functional role of PIWIL3. While 71 the intracellular distributions of PIWIL1 , PIWIL2 and PIWIL4 have been elucidated in mouse 72 testis 10,15 and human fetal oocytes 14 , the subcellular compartment(s) in which PIWIL3 exerts 73 its function remains uninvestigated to date. Herein, we follow the distribution and expression 74 pattern of PIWIL3 in bovine oocytes and preimplantation embryos, by transiently expressing 75 EGFP-PIWIL3 fusion proteins. 76 We generated EGFP-PIWIL3 fusion constructs (both N-and C-terminal tagging), performed in 77 vitro transcription and directly injected EGFP-PIWIL3 mRNA into bovine oocytes at the 78 germinal vesicle (GV) stage and 2 pro-nuclei (2PN) stage zygotes after in vitro fertilization. 79 Zygotes were cultured further in vitro for up to 8 days (blastocyst stage), during which 80 embryos at various developmental stages post fertilization were harvested. 81 First we confirmed that the entire constructs were translated in the oocytes. Microinjection 82 of the EGFP-PIWIL3 and PIWIL3-EGFP fusion construct into oocytes resulted in proteins of 130 83 kDa, corresponding to the total length of EGFP (26 kDa) and PIWIL3 (100 kDa) (Figure 1a).84Next, we followed the localization of EGFP-PIWIL3 in bovine oocytes. Starting from the GV 85 stage, EGFP-PIWIL3 was localized largely to the oocyte cytoplasm in a punctate pattern but 86 was excluded from the nucleus, whereas the EGFP control signal was distributed evenly 87 6 throughout the cell. The expression of PIWIL3 remained cytoplasmic throughout 88 preimplantation development but strongly reduced at the blastocyst stage (F...

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PIWI‐interacting RNAs and human testicular function

Saritas

Main

Winge

et al. 2022

WIREs Mechanisms of Disease

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Small noncoding RNAs (sncRNAs) are pieces of RNA with a length below 200 bp and represent a diverse group of RNAs having many different biological functions. The best described subtype is the microRNAs which primarily function in posttranscriptional gene regulation and appear essential for most physiological processes. Of particular interest for the germline is the PIWI‐interacting RNAs (piRNAs) which are a class of sncRNA of 21–35 bp in length that are almost exclusively found in germ cells. Recently, it has become clear that piRNAs are essential for testicular function, and in this perspective, we outline the current knowledge of piRNAs in humans. Although piRNAs appear unique to germ cells, they have also been described in various somatic cancers and biofluids. Here, we discuss the potential function of piRNAs in somatic tissues and whether detection in biofluids may be used as a biomarker for testicular function. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology

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Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis

Cited by 44 publications

References 49 publications

Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells

Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells

PIWIL3 forms a complex with TDRKH in mammalian oocytes

PIWI‐interacting RNAs and human testicular function

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