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. 2016 Oct 14;44(18):8908-8920.
doi: 10.1093/nar/gkw709. Epub 2016 Aug 11.

Mammalian PNLDC1 is a novel poly(A) specific exonuclease with discrete expression during early development

Dimitrios Anastasakis  1 , Ilias Skeparnias  1 , Athanasios-Nasir Shaukat  1 , Katerina Grafanaki  1 , Alexandra Kanellou  2 , Stavros Taraviras  2 , Dionysios J Papachristou  3 , Athanasios Papakyriakou  4 , Constantinos Stathopoulos  5
Affiliations

Mammalian PNLDC1 is a novel poly(A) specific exonuclease with discrete expression during early development

Dimitrios Anastasakis et al. Nucleic Acids Res. .

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 stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3' RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development.

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Figures

Figure 1.
Figure 1.
(A) Comparison of PNLDC1 and PARN domain architecture. PARN contains a characteristic nuclear localisation signal (NLS, residues 520–540), a carboxy terminal domain (CTD, 540–639) which are not conserved in PNLDC1 and additional regions (represented in yellow) which are absent in PNLDC1. The RRM domain of PARN (445–520) is partially conserved in PNLDC1. Residues 506–525 in HsPNLDC1-1 represent a putative transmembrane domain (TD) (B) Human PNLDC1-1 and PNLDC1-2 exons arrangement. (C) Close-up view of HsPNLDC1 active site model in comparison with the poly(A)-bound X-ray structure of HsPARN nuclease domain (PDB ID: 2A1R). The catalytic DDED-motif residues are illustrated with green C atoms, the conserved and non-conserved RNA-interacting residues are shown with cyan and orange C atoms, respectively. All other atoms are blue for N, red for O and yellow for S. The bound trinucleotide is shown with gray sticks. (D) Autoradiography of in vitro PNLDC1 reaction products analysed as described in the materials and methods section. The reactions took place in the presence of either 5′ end [32P]-labeled N9A15 synthetic RNA substrate or in the presence of 5′ end [32P]-labeled poly(U), poly(C), poly(G), poly(A) and poly(dA) synthetic substrates.
Figure 2.
Figure 2.
Confocal fluorescence microscopy images showing the subcellular localization of EGFP (control), HsPNLDC1-1-EGFP, HsPNLDC1-2-EGFP and MmPNLDC1-EGFP in comparison with the localization of the ER targeted protein mTurquoise-ER in transfected HEK 293 cells. The nuclei were stained with DRAQ5 (blue colour). In order to assist visual interpretation mTurquoise-ER was artificially stained with red colour. The merge channels (with 5 μm scale bar) reveal co-localization of the ER targeted protein and PNLDC1.
Figure 3.
Figure 3.
(A) HEK 293 cells were treated with 100 μM of 5-AZA-CdR for 72 h to achieve maximum inhibitory effect of the promoter methylation. Total RNA was extracted and RT-qPCR was performed to detect expression of PNLDC1 and PARN. The expression of ACTIN was used as a positive control. The products were analysed on a 1.5% agarose gel. Both PNLDC1 isoforms were detected only after demethylation. (B) RT-qPCR results after PARN silencing in HEK 293 cells expressing PNLDC1 after treatment with 5-AZA-CdR for 72 h. PARN knockdown does not affect the expression levels of PNLDC1. (C) Relative expression levels of Pnldc1 among different mouse tissues and during embryogenesis (inset) compared to undifferentiated stem cells. (D) Immunohistochemistry image showing PNLDC1 localization in human testes samples with intratubular germ cell neoplasia. PNLDC1 is detected in the cytoplasm of almost all the neoplastic spermatogenic cells (red arrow). PNLDC1 does not show any immunostaining in the fibromyocytes (yellow arrow) that surrounds the spermatic testicular tubules (original magnification 20X). (E) Immunofluorescence of mouse testes sample. The expression of Pnldc1 is evident in the cells of all the spermatogenic series with higher expression in meiotic spermatocytes (yellow arrows). (F) Immunofluorescence analysis of mouse stem cells. Pnldc1 is shown in red and is localized in the cytoplasm (yellow arrows).
Figure 4.
Figure 4.
(A) Expression levels of Pnldc1 and (B) Dnmt3b, during mESCs differentiation. The expression of Pnldc1 diminishes during the first three days of differentiation. A simultaneous increase of Dnmt3b level is observed during the same period, followed by an equilibrated expression profile until day 7. (C) Effective knockdown of Pnldc1 in mESCs using esiRNA was confirmed by immunofluorescence and (D) RT-qPCR (E) Expression pattern of Cnot7, Cnot6l, Cnot6, Cnot8, Pan2, Parn and Angel analysed by RT-qPCR after Pnldc1 knockdown. (*P value < 0.05, **P value < 0.01; ***P value < 0.001).
Figure 5.
Figure 5.
Classes of biological processes and molecular functions related to the genes with significant differential expression in mESCs after Pnldc1 knockdown. Gene ontology enrichment analysis was performed using the PANTHER classification system as described and P-values were subjected to Bonferroni correction for multiple testing.
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