SNORD88B promotes proliferation and invasion of trophoblast by regulating G3BP1 m6 A modification and alternative splicing

Abstract

Background: Small nucleolar RNA (snoRNA) has recently been shown to play a key role in different diseases, but how snoRNA works to promote the biological behavior of trophoblast remains unclear.

Methods: The expression of snoRNA in the placental villi of females were detected by RNA-seq and qPCR. RNA immunoprecipitation (RIP), RNA methylation co-immunoprecipitation (Me-RIP), and cell experiments were used to explore the molecular mechanism of SNORD88B in regulating the function of villous trophoblasts.

Results: SNORD88B was down-regulated in the placental villi of patients with spontaneous abortion, while the overexpression of SNORD88B could promote the proliferation, invasion, and migration of villous trophoblast cells. Mechanistically, SNORD88B directly binds to m6A methyltransferase METTL14 to regulate the m6A modification of G3BP1 mRNA and enhances its mRNA stability by recruiting the reader IGF2BP2. Besides, SNORD88B also regulates the alternative splicing of pre-G3BP1 by recruiting splicing factors SRSF1 and U2AF1, which ameliorates the expression of its effective transcripts to further promote the expression of G3BP1 protein.

Conclusions: We investigated the functional roles and the underlying regulatory mechanisms of SNORD88B in the enhancement of villous trophoblasts' development and that under-expressed SNORD88B could contribute to the maldevelopment of placental villi, which may be related to spontaneous abortion.

Keywords: Alternative splicing; Early pregnancy loss; G3BP1; M6 A modification; SNORD88B.

Fig. 1

SNORD88B is down-regulated in placental villi of SA patients. RNA-seq analysis shows the abnormal expression of snoRNAs in placental villi of patients with spontaneous abortion (SA) compared with normal pregnancy (NP) (A&B). The Venn plot shows the downregulated expression of snoRNAs in our dataset and GSE123719 dataset in SA, with only three snoRNAs downregulated in both datasets (C). QPCR assay showed that only SNORD88B was down-regulated in placental villi of patients with spontaneous abortion (n = 47), compared with placental villi of normal pregnancy (n = 23) (D-F). Nucleoplasm separation assay show that SNORD88B is mainly distributed in the nucleus (97.5%), and a small part is located in the cytoplasm (2.5%) (G). Immunofluorescence showed that SNORD88B was located predominantly in the nucleus, with a small amount distributed in the cytoplasm (H). Secondary structure analysis reveals that SNORD88B has a classical snoRNA stem-loop structure (I). ns means P > 0.05, *P < 0.05, (assessed using Student’s t-test)

Fig. 2

SNORD88B promotes proliferation, invasion and migration of villous trophoblast cells. ASO targeting SNORD88B was used to knock down the expression level of SNORD88B in HTR-8/SVneo and JEG-3 cells (A&B). Transfection of SNORD88B overexpression plasmid in HTR-8/SVneo cells increases the expression level of SNORD88B (C). Knockdown of SNORD88B inhibited cell proliferation (D&E), induced apoptosis (F) inhibited cell migration (G) and invasion (H) in HTR-8/SVneo and JEG-3 cell lines. Overexpression of SNORD88B accelerated cell proliferation (I), reduced apoptosis (J), increased migration and invasion (K) in HTR-8/SVneo cells. *P < 0.05 (assessed using One-way ANOVA or Student’s t-test)

Fig. 3

G3BP1 is a direct downstream target of SNORD88B in trophoblasts. NCBI blast analysis showed that there is a 14 bp complementary region between SNORD88B and G3BP1 (A). Knockdown of SNORD88B decreased G3BP1 mRNA level while overexpression of SNORD88B increased G3BP1 mRNA level (B). Knockdown of SNORD88B decreased G3BP1 protein level while overexpressing of SNORD88B increased G3BP1 protein level (C). Detection of the expression correlation between SNORD88B and G3BP1 in human placental villus tissue (D). Knockdown of G3BP1 in SNORD88B-overexpressing cells reversed the effects of SNORD88B on trophoblast proliferation (E), apoptosis (F), migration (G) and invasion (H). *P < 0.05 (assessed using One-way ANOVA or Student’s t-test)

Fig. 4

SNORD88B activates EMT and Wnt pathways by upregulating G3BP1. Knockdown of SNORD88B increased E-cadherin, reduced N-cadherin, increased GSK-3β and reduced β-catenin proteins level in HTR-8-SVneo and JEG-3 cells while overexpressing of SNORD88B decreased E-cadherin, increased N-cadherin, decreased GSK-3β and increased β-catenin proteins (A). Knockdown of G3BP1 in SNORD88B-overexpressing cells reversed the effect of SNORD88B on E-cadherin, N-cadherin, GSK-3β and β-catenin (B). Knockdown of FBL in SNORD88B-overexpressing cells did not affect the protein level of G3BP1 (C). RIP assay showed that SNORD88B did not bind to FBL (D). ns means P > 0.05, *P < 0.05 (assessed using Student’s t-test)

Fig. 5

SNORD88B enhances G3BP1 mRNA stability by regulating G3BP1 m6 A modification. Intersection of catRAPID and ENCORI database reveals 22 RNA-binding proteins that may bind SNORD88B (A). GO enrichment analysis showed that these RBPs are closely related to RNA processing (B), and further analysis showed that these proteins are mainly related to m6 A modification and pre-mRNA splicing. m6 A dot blot and immunofluorescence assay showed that overexpression of SNORD88B in HTR-8/SVneo cells significantly increased the overall m6 A modification level, and downregulation of SNORD88B in JEG-3 cells decreased the overall m6 A level (C&D). The SRAMP database predicts that there are multiple high-confidence m6 A modification sites in G3BP1 mRNA (E). Overexpression of SNORD88B in HTR-8/SVneo cells significantly increased the m6 A modification level of G3BP1 (F). Downregulation of SNORD88B in JEG-3 cells decreased the m6 A modification level of G3BP1 (G). RIP assay showed that m6 A methyltransferase METTL14 simultaneously bound to SNORD88B and G3BP1 mRNA, while the m6 A reader with the highest binding level to SNORD88B and G3BP1 mRNA was IGF2BP2 (H-K). Knockdown of METTL14 in SNORD88B-overexpressing cells significantly decreased the level of G3BP1 m6 A modification (L). Knockdown of SNORD88B in HTR-8/SVneo and JEG-3 cells resulted in reduced stability and accelerated decay of G3BP1 mRNA (M&N), while Knockdown of IGF2BP2 in SNORD88B- overexpressing cells reversed the effect of SNORD88B on G3BP1 mRNA stability (O). Knockdown of METTL14 or IGF2BP2 in SNORD88B-overexpressing cells reversed the effects of SNORD88B on trophoblast proliferation (P), apoptosis (Q), migration and invasion (R). Knockdown of METTL14 or IGF2BP2 in SNORD88B-overexpressing cells reversed the effects of SNORD88B on E-cadherin, N-cadherin, GSK-3β and β-catenin (S). *P < 0.05 (assessed using One-way ANOVA or Student’s t-test)

Fig. 6

SNORD88B regulates pre-G3BP1 alternative splicing and increases G3BP1 effective transcript expression via recruiting SRSF1 and U2 AF1. G3BP1-associated transcript isoforms, including wild-type, exon skipping, and intron retention (A). QPCR and DNA electrophoresis assay showed that knockdown of SNORD88B would lead to increased levels of two nonsense transcripts (G3BP1_X1, G3BP1_X2) (B-D). RIP assay showed that splicing regulator SRSF1 and U2 AF1 can simultaneously bound SNORD88B and pre-G3BP1, and had the highest binding abundance (E-H). Knockdown of SRSF1 or U2 AF1 in SNORD88B-overexpressing cells resulted in increased pre-G3BP1 and decreased G3BP1-wild-type transcript (I&J), and increased the level of nonsense transcripts G3BP1_X1 and G3BP1_X2 (K). Knockdown of SRSF1 or U2 AF1 in SNORD88B-overexpressing cells reversed the effects of SNORD88B on trophoblast proliferation (L), apoptosis, migration and invasion (M). Knockdown of SRSF1 or U2 AF1 in SNORD88B-overexpressing cells reversed the effects of SNORD88B on E-cadherin, N-cadherin, GSK-3β and β-catenin (N). ns means P > 0.05, *P < 0.05 (assessed using One-way ANOVA or Student’s t-test)

Fig. 7

ASO-mediated downregulation of SNORD88B results in functional defects in extravillous explants. Extravillous explants from NP and SA patients were maintained in culture on Matrigel. Downregulation of SNORD88B results in a significant reduction in villus explant migration distance (A). Knockdown of SNORD88B results in decreased G3BP1 expression in extravillous explants (B). *P < 0.05 (assessed using Student’s t-test)