Abstract:PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic … Show more
“…We propose that in bovine oocytes, TDRKH is also important for PIWIL3 recruitment to mitochondria since PIWIL3 physically interacts with TDRKH and co-localizes with TDRKH on the mitochondria. This TDRKH binding brings the piRNA-bound PIWIL3 in close proximity to PNLDC1, a deadenylase responsible for the elimination of mRNA 3’ poly(A) tails and trimming during pre-pachytene and pachytene pre-piRNA maturation 25,30,31 .…”
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...
“…We propose that in bovine oocytes, TDRKH is also important for PIWIL3 recruitment to mitochondria since PIWIL3 physically interacts with TDRKH and co-localizes with TDRKH on the mitochondria. This TDRKH binding brings the piRNA-bound PIWIL3 in close proximity to PNLDC1, a deadenylase responsible for the elimination of mRNA 3’ poly(A) tails and trimming during pre-pachytene and pachytene pre-piRNA maturation 25,30,31 .…”
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...
“…2). The unique domains of each protein likely mediate subcellular localization, substrate, and regulatory partner specificity, as has been observed for CAF1z [30,35,36], PDE12 [37–39], PARN [36,40–42], and PNLDC1 [43–46]. NOCT biological function is likely further defined by its expression pattern and subcellular localization.10.1080/15476286.2018.1526541-F0002Figure 2.NOCT is related to CCR4-type deadenylases and contains a conserved EEP catalytic domain.…”
Section: Post-transcriptional Control Of Mrna Abundance and Stabilitymentioning
confidence: 99%
“…Several deadenylases exhibit specific subcellular distribution [35,36–38,40–43], raising the question of whether NOCT activity may be spatially restricted. Studies of overexpressed tagged constructs found Xenopus NOCT to be cytoplasmic in photoreceptor cells [95], while mouse NOCT is perinuclear in human HEK293 cells [57] and is excluded from stress granules in mouse NIH3T3 cells [56].…”
Section: Is Noct Activity Spatially Restricted?mentioning
Post-transcriptional control of messenger RNA (mRNA) is an important layer of gene regulation that modulates mRNA decay, translation, and localization. Eukaryotic mRNA decay begins with the catalytic removal of the 3′ poly-adenosine tail by deadenylase enzymes. Multiple deadenylases have been identified in vertebrates and are known to have distinct biological roles; among these proteins is Nocturnin, which has been linked to circadian biology, adipogenesis, osteogenesis, and obesity. Multiple studies have investigated Nocturnin’s involvement in these processes; however, a full understanding of its molecular function remains elusive. Recent studies have provided new insights by identifying putative Nocturnin-regulated mRNAs in mice and by determining the structure and regulatory activities of human Nocturnin. This review seeks to integrate these new discoveries into our understanding of Nocturnin’s regulatory functions and highlight the important remaining unanswered questions surrounding its regulation, biochemical activities, protein partners, and target mRNAs.
“…Mass spectrometric analysis of protein components within the Papi complex immunoprecipiated from the cultured Bombyx cells led to the identification of a homolog of PNLDC1, termed Trim, as the piRNA 3′ processing factor (Figure B) . Trim is composed of CAF1 family ribonuclease and TM domains (Table ) through which it is localized on the mitochondrial surface, as is Papi.…”
Section: Bombyx Pirna Factors and Their Functionsmentioning
The PIWI-interacting RNA (piRNA) pathway, one of the major eukaryotic small RNA silencing pathways, is a genome surveillance system that silences selfish genes in animal gonads. piRNAs guide PIWI protein to target genes through Watson-Crick RNA-RNA base-parings. Loss of piRNA function causes genome instability, inducing failure in gametogenesis and infertility. Studies using fruit flies and mice as key experimental models have resulted in tremendous progress in understanding the mechanism underlying the piRNA pathway. Recent work using cultured silkworm germline cells has also expanded our knowledge of piRNA biogenesis in particular, since these silkworm cells are the only cells of germline origin that can be cultured. In this review, we describe elucidation of the piRNA pathway using cultured silkworm cells as an experimental model by focusing on recent work in biochemistry and structural biology. Earlier studies that made important contributions to the field are also described.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
