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, 37 (2), 507-523

Eutherian-Specific Gene TRIML2 Attenuates Inflammation in the Evolution of Placentation


Eutherian-Specific Gene TRIML2 Attenuates Inflammation in the Evolution of Placentation

Xuzhe Zhang et al. Mol Biol Evol.


Evolution of highly invasive placentation in the stem lineage of eutherians and subsequent extension of pregnancy set eutherians apart from other mammals, that is, marsupials with short-lived placentas, and oviparous monotremes. Recent studies suggest that eutherian implantation evolved from marsupial attachment reaction, an inflammatory process induced by the direct contact of fetal placenta with maternal endometrium after the breakdown of the shell coat, and shortly before the onset of parturition. Unique to eutherians, a dramatic downregulation of inflammation after implantation prevents the onset of premature parturition, and is critical for the maintenance of gestation. This downregulation likely involved evolutionary changes on maternal as well as fetal/placental side. Tripartite-motif family-like2 (TRIML2) only exists in eutherian genomes and shows preferential expression in preimplantation embryos, and trophoblast-derived structures, such as chorion and placental disc. Comparative genomic evidence supports that TRIML2 originated from a gene duplication event in the stem lineage of Eutheria that also gave rise to eutherian TRIML1. Compared with TRIML1, TRIML2 lost the catalytic RING domain of E3 ligase. However, only TRIML2 is induced in human choriocarcinoma cell line JEG3 with poly(I:C) treatment to simulate inflammation during viral infection. Its knockdown increases the production of proinflammatory cytokines and reduces trophoblast survival during poly(I:C) stimulation, while its overexpression reduces proinflammatory cytokine production, supporting TRIML2's role as a regulatory inhibitor of the inflammatory pathways in trophoblasts. TRIML2's potential virus-interacting PRY/SPRY domain shows significant signature of selection, suggesting its contribution to the evolution of eutherian-specific inflammation regulation during placentation.

Keywords: Eutheria; TRIM family protein; coevolution; endogenous retrovirus; inflammation; placentation.


<sc>Fig</sc>. 1.
Fig. 1.
TRIML2 likely resulted from a gene duplication event in eutherian stem lineage. (A) Human TRIML1 and TRIML2 are located at the telomeric end of the long arm of chromosome 4. (B and C) Multiple alignments of TRIML1 and TRIML2 loci show both genes exist in all four superorders of eutherian. (D–G) Phylogenetic trees generated through maximum likelihood (D, mRNA tree; F, protein tree) and Bayesian analyses (E, mRNA tree; G, protein tree), with bootstrap percentages/posterior probabilities.
<sc>Fig</sc>. 2.
Fig. 2.
TRIML1 and TRIML2 show different restricted expression patterns. (A) Heatmap of relative tissue expression levels of human TRIM genes, showing restricted expression of TRIML1 and TRIML2 (data from the Human Protein Atlas, expression level in each tissue normalized to the highest RPKM for each TRIM gene, supplementary fig. S4, Supplementary Material online). (B) Preferential expression compartment of TRIML1, TRIML2, and ZFP42 in extraembryonic tissues correlates with their transcribing direction. Expression levels normalized to placenta villi. Values are mean ± SEM. One-way ANOVA, Tukey’s HSD, **P < 0.01, *P < 0.05. Deci, placental decidua; Vill, placental villi; Chor, chorion; Amni, amnion; Cen, centromere; 4qter, terminal of chromosome 4 long arm; arrows indicate the transcription direction of each gene. (C) Triml1 and Triml2 expression in adult and embryo mouse tissue show restricted expression in preimplantation embryo, extraembryonic tissues, testis, and ovary. Expression levels normalized to placental villi. N.D., not detected; 2-Cell, 2-cell stage embryo; 8-Cell, 8-cell stage embryo; Blasto, blastocyst; Lab, E15.5 labyrinth; Ju+De, E15.5 junctional zone, and decidua; Mem, E15.5 fetal membranes; E-Brain, E15.5 embryo brain; E-Limb, E15.5 embryo limb; E-Liver, E15.5 embryo liver; E-Lung, E15.5 embryo lung. (D) Triml1 and Triml2 show higher expression in fetal membrane than in placental labyrinth. Expression levels normalized to E19.0 labyrinth. Values are mean ± SEM. t-Test, ***P < 0.001, **P < 0.01, *P < 0.05. (E) RNAScope assay showing Triml1 and Triml2 expression in extraembryonic structures, upper row: signal from Triml1 and Triml2-specific probes, lower row: nonspecific signals from negative control probes.
<sc>Fig</sc>. 3.
Fig. 3.
TRIML1 and TRIML2 expression during trophoblast differentiation. (A and B) TRIML1 and TRIML2 expression differ during forskolin-induced BeWo cell syncytialization. (C and D) GSK3ixv-induced mouse TSCs differentiation into syncytiotrophoblast layer II causes downregulation of both Triml1 and Triml2. Expression levels normalized to control groups. Values are mean ± SEM. One-way ANOVA, Tukey’s HSD, ***P < 0.001, **P < 0.01, *P < 0.05. n.s., not statistically significant.
<sc>Fig</sc>. 4.
Fig. 4.
TRIML2 during trophoblast inflammation. (A and B) TRIML2 expression in JEG3 is induced by poly(I:C). (C–E) TRIML2 knockdown during inflammation causes increased expression of proinflammatory cytokines IFNB1 (C) and IL6 (D), and decreased survival of JEG3 cells (E). TRIML2 overexpression reduces IFNB1 (F) and IL6 (G) expression at baseline. Normalized to PBS-treated control groups; IC, poly(I:C), OE, overexpression. Values are mean ± SEM. (A, B, F, and G), t-test. (C and D) One-way ANOVA, Tukey’s HSD. Poly(I:C) treatment in (E), Kruskal–Wallis test, Bonferroni post hoc correction. ***P < 0.001, **P < 0.01, *P < 0.05. n.s., not statistically significant.
<sc>Fig</sc>. 5.
Fig. 5.
Sites under positive selection in TRIML1 and TRIML2. (A) Compared with monotreme and marsupial TRIML1, eutherian TRIML1 lacks the B-box domain, while TRIML2 lacks both the RING and the B-box domains. In TRIML1, the site under significant positive selection resides in B-box homologous region. In TRIML2, sites under significant positive selection concentrate in B-box homologous region, coiled-coil domain and PRY/SPRY domain. (B) Alignment of human TRIML2 and TRIM5. Positively selected sites (red) cluster in the specificity-determining v2 region (orange shade) and are flanked by conserved sites (green) that contribute to the hydrophobic core of TRIM’s canonical binding interface.

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