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M-RAS Regulate CDH1 Function in Blastomere Compaction during Porcine Embryonic Development

M-Ras shares ∼50% sequence identity with the canonical Ras proteins (Kimmelman et al., 1997), and, although it can interact with some of the well-established Ras effectors, such as Raf, PI3K, and RalGDS. M-Ras also binds to distinct effectors, including certain RapGEFs and the Shoc2 scaffold (Herrmann et al., 1996; Rebhun et al., 2000; Gao et al., 2001; Rodriguez-Viciana et al., 2006). In contrast to the canonical Ras proteins, constitutively active M-Ras has a low transforming activity and is rarely mutated in human cancer (Quilliam et al., 1999); however, gain-of-function mutations in M-RAS have been identified in patients with Noonan syndrome (Higgins et al., 2017). Notably, mutations in one of the distinct effectors of M-RAS, the SHOC2 scaffold, have also been detected in Noonan patients (Cordeddu and Di, 2009; Hannig et al., 2014), and, likewise, Shoc2 has been implicated in cell migratory events (Young et al., 2013; Kaduwal et al., 2015; Jeoung et al., 2016). Subsequent biochemical studies have shown that the direct binding of Shoc2 to active GTP-bound M-Ras allows the Shoc2 scaffold to nucleate a ternary complex consisting of active M-Ras, Shoc2 and the catalytic subunit of PP1 (PP1c) (Rodriguez-Viciana et al., 2006). In RTKmediated signaling, the M-Ras/Shoc2/PP1c ternary comM-RAS Regulate CDH1 Function in Blastomere Compaction during Porcine Embryonic Development

In contrast to the canonical Ras proteins, constitutively active M-Ras has a low transforming activity and is rarely mutated in human cancer (Quilliam et al., 1999); however, gain-of-function mutations in M-RAS have been iden-tified in patients with Noonan syndrome (Higgins et al., 2017). Notably, mutations in one of the distinct effectors of M-RAS, the SHOC2 scaffold, have also been detected in Noonan patients (Cordeddu and Di, 2009;Hannig et al., 2014), and, likewise, Shoc2 has been implicated in cell migratory events (Young et al., 2013;Kaduwal et al., 2015;Jeoung et al., 2016).
Subsequent biochemical studies have shown that the direct binding of Shoc2 to active GTP-bound M-Ras allows the Shoc2 scaffold to nucleate a ternary complex consisting of active M-Ras, Shoc2 and the catalytic subunit of PP1 (PP1c) (Rodriguez-Viciana et al., 2006). In RTKmediated signaling, the M-Ras/Shoc2/PP1c ternary com-plex functions to dephosphorylate a negative regulatory 14-3-3 binding site on the Raf kinases, which promotes Raf binding to the canonical Ras proteins and facilitates ERK cascade activation (Rodriguez-Viciana et al., 2006;Matsunaga-Udagawa et al., 2010;Galperin et al., 2012). This complex has been implicated in the dynamic regulation of ERK activity and cell polarity in some cancer cell lines (Young et al., 2013). Moreover, M-Ras/Shoc2 signaling contributes to junction turnover by modulating the Cdh1/p120-catenin interaction and, in turn, the junctional expression of Cdh1. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphor-regulation of p120-catenin and required downstream ERK cascade activation (Kota et al., 2019).
Compaction appears to occur in all mammalian embryos but at different cell cycles. In the pig, compaction and cell polarization occur more gradually than in the mouse and coincide with relocation of CDH1 to contact sites, the process not completing long before blastocyst formation (Reima et al., 1993). In the developing embryo CDH1 is a key player during morula compaction and CDH1 null embryos die early during embryogenesis as development of the blastocyst and trophectoderm is impaired (Larue et al., 1994;Riethmacher et al., 1995).
To further elucidate the biological functions of the M-RAS on porcine embryonic compaction, we investigated the mechanism by which M-RAS and CDH1 contribute to the regulation of compaction.

Reagents
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Oocyte collection and in vitro maturation (IVM)
All animal studies were performed in strict accordance with the institutional guideline and prior approval from the Institutional Animal Care and Use Committee (IACUC) of the Chungbuk National University, Republic of Korea.

Parthenogenetic activation and in vitro culture (IVC)
For parthenogenetic activation, mature oocytes were

M-RAS double-stranded RNA preparation
To prepare M-RAS double-stranded RNA (dsRNA), M-RAS was amplified using a pair of primers (Table 1) containing the T7 promoter sequence. The purified PCR products were then used to synthesize dsRNA with the MEGAscript T7 Kit (AM1333; Thermo Fisher Scientific) according to the manufacturer's instructions. After in vitro transcription, dsRNA was treated with DNase I and RNase A to remove the DNA template and any single-stranded RNAs, followed by purification with phenol-chloroform extraction and isopropyl alcohol precipitation. The puri-fied dsRNA was dissolved in RNase-free water and stored at -80 o C until use.

Cloning and in vitro mRNA synthesis
The open reading frames of pig M-RAS (Table 1)

Immunofluorescence and confocal microscopy
After washing 3 times with PBS and PVA, embryos were fixed in 3.7% paraformaldehyde for 30 min at room temperature, permeabilized with PBS and PVA contain-

Expression patterns of M-RAS during porcine embryo development
We evaluated the expression and subcellular location of M-RAS at different stages during porcine embryo development (Fig. 1). M-RAS was expressed in the cytoplasm at zygote stage and highly expressed on the membrane from 2-cell stage until morula stage. The signal was detected on the nucleus from blastocyst (Fig. 1A). Moreover, the protein expression was decreased during preimplanta-

A B
Zygote 2  tion development (Fig. 1B). The mRNA expression level of M-RAS was detected by qPCR. M-RAS was maternally expressed in MII oocytes. Thereafter, M-RAS expression decreased and remained at a low level until blastocyst (Fig.   1C).

Effects of M-RAS knockdown on early porcine embryonic development
To western blotting (Fig. 2E).

The active M-RAS regulates cell-cell adhesion by modulating the E-cadherin which correlates with ERK cascade activation
To more directly assess the role of M-RAS in regulating intercellular adhesion, we examined whether the presence or absence of M-RAS had any effects on E-cadherin localization in different stages of embryos (Fig. 3). In M-RAS KD embryos, the decrease in cell-cell adhesion coincided with a lower expression of E-cadherin shown by immune fluorescence and western blotting (Fig. 3A, 3B).
However, no detectable change in ZO-1 (also known as tight junction protein-1) was present in M-RAS KD porcine embryos (Fig. 3C). In addition, the phosphorylation level of ERK was significantly higher than M-RAS KD embryos.

Overexpression of M-RAS rescues the disrupt of embryonic compaction after M-RAS knockdown
To confirm that the role of the M-RAS on embryonic compaction, we first produced the cRNA of activated M-   et al., 2005;Schubbert et al., 2006;Cirstea and Kdvorsky, 2010;Aoki et al., 2013;Flex et al., 2014).   Elevated expression of E-cadherin at adhesion junctions is known to increase cell-cell adhesiveness, and binding of p120-catenin to the juxta membrane domain of E-cadherin promotes the surface stability of E-cadherin by preventing its endocytosis and degradation. In particular, we found that E-CADHEREN protein level of M-RAS KD embryos was reduced whereas interaction between E-cadherin and p120-catenin previously reported to increase cell-cell adhesiveness was increased (Kota et al., 2019). In this study, we demonstrate that M-RAS transduces signal specific to the embryonic program including preimplantation development and compaction.

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.