Origination of LTR Retroelement-Derived NYNRIN Coincides with Therian Placental Emergence

Abstract

The emergence of the placenta is a revolutionary event in the evolution of therian mammals, to which some LTR retroelement-derived genes, such as PEG10, RTL1, and syncytin, are known to contribute. However, therian genomes contain many more LTR retroelement-derived genes that may also have contributed to placental evolution. We conducted large-scale evolutionary genomic and transcriptomic analyses to comprehensively search for LTR retroelement-derived genes whose origination coincided with therian placental emergence and that became consistently expressed in therian placentae. We identified NYNRIN as another Ty3/Gypsy LTR retroelement-derived gene likely to contribute to placental emergence in the therian stem lineage. NYNRIN knockdown inhibited the invasion of HTR8/SVneo invasive-type trophoblasts, whereas the knockdown of its nonretroelement-derived homolog KHNYN did not. Functional enrichment analyses suggested that NYNRIN modulates trophoblast invasion by regulating epithelial-mesenchymal transition and extracellular matrix remodeling and that the ubiquitin-proteasome system is responsible for the functional differences between NYNRIN and KHNYN. These findings extend our knowledge of the roles of LTR retroelement-derived genes in the evolution of therian mammals.

Keywords: LTR retrotransposon; ancestral state reconstruction; evolutionary genomics; placenta.

Fig. 1.

Many LTR retroelement–derived genes could be involved in therian placental evolution. (A) Schematic differentiation of trophoblast-lineage cells in the tammar wallaby (nearly full-term) and human (third trimester) placentae from blastocyst trophectoderm cells, as examples of the differentiation in marsupials and that in placentals, respectively. (B) A simplified phylogeny and the numbers of tetrapod genomes or transcriptomes that were used in this study. (C and D) Schematic representations of transition events regarding (C) gene sequence and expression gain or loss or (D) integration of LTR retroelements. (E) A schematic diagram showing the reconstruction of ancestral states of genes and interpretation of transition events from character state changes. (F) Numbers of identified ortholog groups whose ancestral gene originated in the stem lineage of each clade but was not expressed in the placenta or chorioallantoic membrane of its MRCA (state 0 → 1) and those whose ancestral gene originated in the stem lineage of the MRCA and became expressed in the placenta or chorioallantoic membrane of that MRCA (state 0 → 2). (G) Numbers of identified ortholog groups whose ancestral gene in the stem lineage of each clade was newly integrated by at least an LTR retroelement, and all the integrated LTR retroelements became introns (state A → I) or at least an LTR retroelement became an exon (state A → E) in the gene of the MRCA of that clade. (H) Numbers of ortholog groups of LTR retroelement–derived genes that may have contributed to the placental evolution in the therian, marsupial, and placental lineages (states 0 → 2 and A → E).

Fig. 2.

NYNRIN originated in the therian stem lineage and its expression was recruited into the placenta of the therian MRCA. (A and B) Reconstruction of the ancestral states of NYNRIN in terms of (A) the presence/absence of gene sequences and their expression in the placenta or chorioallantoic membrane or (B) the presence/absence of LTR retroelements that became exons of NYNRIN. Marginal posterior probability approximation method was used. Node color represents the estimated states. Blue arrow: species whose transcriptome data from the placenta or chorioallantoic membrane were available, crescent: mammalian MRCA, star: therian MRCA. (C) Domain architecture of the human NYNRIN, KHNYN, and other phylogenetically related NYN-containing N4BP1, ZC3H12C, and ZC3H12A proteins. KH-like, K homology-like domain; NYN, Nedd4-BP1 YacP nuclease domain; INT, integrase domain; CUBAN, cullin-binding domain associating with NEDD8 domain; CoCUN, cousin of CUBAN domain; UBA, ubiquitin-associated domain.

Fig. 3.

NYNRIN promotes trophoblast invasion rather than trophoblast fusion. (A) Human placental tissues from a healthy individual and those from a patient with preeclampsia and intrauterine growth restriction (IUGR) stained with anti-NYNRIN, anti-HLA-G (a marker of extravillous trophoblast), or normal rabbit IgG (negative control) (20X). ET (red arrow): extravillous trophoblast in decidual tissue, CT (yellow arrow): cytotrophoblast, ST (blue arrow): syncytiotrophoblast layer. (B) BeWo cells treated with siCtrl (control), siCtrl + Db, or siNYNRIN3 + Db (20×). Blue: nuclei, red: cell membranes, white arrow: fused syncytium. (C) Microscopic images (10×) and (D) proportions of HTR8/SVneo cells that invaded through Matrigel after treatment with siCtrl or each of the three siNYNRINs (siNYNRIN2 for the microscopic image). Blue: nuclei, *P < 0.05, **P < 0.01, ****P < 0.0001, orange lines: 95% confidence intervals for the median.

Fig. 4.

NYNRIN promotes trophoblast invasion by regulating genes related to epithelial-mesenchymal transition and extracellular matrix remodeling. (A) Principal component plot of gene expression profiles of HTR8/SVneo cells treated with siCtrl or siNYNRIN. (B) Volcano plot showing negative log adjusted P-values from DESeq2 and log₂ fold change of genes in NYNRIN-KD HTR8/SVneo cells. The top ten most significantly downregulated or upregulated DEGs are indicated. (C–G) Functional enrichment plots showing gene ratios and adjusted P-values, from clusterProfiler, of the top ten (C) GO Biological Process, (D) GO Molecular Function, (E) GO Cellular Component, (F) KEGG, or (G) MGI Mammalian Phenotype terms enriched with downregulated or upregulated genes among the top 1,000 most significant DEGs in NYNRIN-KD HTR8/SVneo cells. The number in parentheses indicates the number of DEGs that have annotations in each functional database. (H) Heatmap showing the differences in expression of selected genes in NYNRIN-KD (siNYNRYN) and KHNYN-KD (siKHNYN) HTR8/SVneo cells against the expression in the control cells. EpiM, epithelial marker genes; MesM, mesenchymal marker genes; MMP, matrix metalloproteinase; PLAU, plasminogen; IL, interleukin.

Fig. 5.

NYNRIN and KHNYN regulated trophoblast invasion differently. (A) Microscopic images (10×) and (B) proportions of HTR8/SVneo cells that invaded through Matrigel after treatment with siCtrl, siNYNRIN, siKHNYN, or both siNYNRIN and siKHNYN. Blue: nuclei, ns: nonsignificant, *P < 0.05, **P < 0.01, orange lines: 95% confidence intervals for the median. (C) Principal component plot of gene expression profiles of HTR8/SVneo cells treated with siCtrl, siNYNRIN, or siKHNYN. (D) Rank-rank hypergeometric overlap plot showing the degrees of concordance/discordance between upregulated and downregulated genes in NYNRIN-KD and KHNYN-KD HTR8/SVneo cells (negative log P-values). (E) Scatter plot showing log₂ fold change of the top 1,000 genes with the highest disco scores (Domaszewska et al. 2017) whose expression was upregulated or downregulated by either NYNRIN KD or KHNYN KD compared with the control. N-D/K-D, N-U/K-U, N-D/K-UX, and N-U/K-DX denote gene groups with different expression regulation patterns (e.g., N-D/K-UX indicates the group of genes whose expression was downregulated in NYNRIN-KD but upregulated or unchanged in KHNYN-KD). (F–J) Functional enrichment plots showing gene ratios and adjusted P-values, from clusterProfiler, of the top ten (F) GO Biological Process, (G) GO Molecular Function, (H) GO Cellular Component, (I) KEGG, or (J) MGI Mammalian Phenotype terms enriched with the top 1,000 genes with the highest disco scores shown in (E). The number in parentheses represents the number of DEGs that have annotations in each functional database.

Fig. 6.

Roles of NYNRIN in promoting trophoblast invasion could be derived from its putative ubiquitin-binding domain, but not its NYN domain. (A) A predicted 3D structure of human NYNRIN (see Materials and Methods). The circle indicates where the INT domain connects the KH-like and NYN domains with hydrogen bonds. Purple: KH-like domain, light blue: NYN domain, blue: RNase H domain, red: INT domain, orange: hypothetical LTR retroelement–derived segment excluding the RNase H and INT domains, light green: hypothetical nonretroelement-derived segment excluding the KH-like and NYN domains. (B) 3D superimposition of NYN domains of NYNRIN and KHNYN showing mutations in DDDD motifs. Light blue: NYNRIN, light green: KHNYN, blue dashed lines: hydrogen bonds. (C) Predicted 3D structures of KHNYN CUBAN (top) and the region of NYNRIN aligned with KHNYN CUBAN (bottom). The aligned regions were colored by the rainbow rule, from blue on the N-terminus to red on the C-terminus. Numbers represent the order of ɑ-helices along the proteins.