5-Methylcytosine (m5C) is one of the most prevalent internal modifications of messenger RNA (mRNA) in higher eukaryotes. Here we report that Y box protein 2 (YBX2) serves as a novel mammalian m5C binding protein to undergo liquid-liquid phase separation (LLPS) both in vivo and in vitro, and this YBX2-dependent LLPS is enhanced by m5C marked RNA. Furthermore, the crystal structure assay revealed that W100, as a distinct m5C binding site of YBX2, is critical in mediating YBX2 phase separation. Our study resolved the relationship between RNA m5C and phase separation, providing a clue for a new regulatory layer of epigenetics.

RNA-binding proteins (RBPs) fine-tune gene expression by modulating RNA stability, translation, and degradation. RBPs are involved in the development of endometrial cancer. In particular, Y-box-binding protein 2 (YBX2), a germ cell-specific member of the YBX family, has been reported to maintain cancer stem cell-like phenotypes in endometrial cancer. However, the mechanism by which YBX2 modulates mRNA stability in endometrial cancer cells remains unknown. In this study, we examined the effects of the ectopic expression of YBX2 in endometrial adenocarcinoma-derived Ishikawa cells. We found that elevated levels of YBX2 delayed cell proliferation, without increasing cell apoptosis. Transcriptomic analysis revealed disturbances in gene expression caused by YBX2. Interestingly, heat shock protein family A (Hsp70) member 6 (HSPA6) levels were downregulated due to the reduced mRNA stability after YBX2 binding. YBX2 facilitated the formation of relatively stable cytoplasmic granules in tumor cells via its mRNA binding domain. Moreover, N6-methyladenosine (m6A) reader proteins are recruited by YBX2 granules via the cold-shock domain. Notably, knockdown of YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2), an m6A reader, ameliorated the reduction in HSPA6 mRNA levels induced by YBX2, indicating the synergistic effects of YBX2 and YTHDF2 on mRNA stability. Therefore, YBX2 regulates RNA stability by interacting with the m6A reader proteins.

Motile cilia in a vertebrate are important to sustaining activities of life. Fluid flow on the apical surface of several tissues, including bronchial epithelium, ependymal epithelium, and fallopian tubules is generated by the ciliary beating of motile cilia. Multi-ciliated cells in ependymal tissue are responsible for the circulation of cerebrospinal fluid (CSF), which is essential for the development and homeostasis of the central nervous system, and airway tissues are protected from external contaminants by cilia-driven mucosal flow over the top of the airway epithelium.
A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating.
Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia.
The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia.
Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.

Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.

BNC1 is a transcription factor that is crucial for spermatogenesis and male fertility, although the underlying mechanism remains unclear. To study BNC1\'s specific role in spermatogenesis, we characterized a previously developed mouse model carrying a truncating mutation in Bnc1 (termed Bnc1+/tr for heterozygotes and Bnc1tr/tr for homozygotes) and found that the mutation decreased BNC1 protein levels and resulted in germ cell loss by apoptosis. Given that loss of functional Bnc1 is known to result in decreased expression of the spermatogenesis genes Ybx2 and Papolb, we aimed to explore whether and how BNC1 promotes transcription of Ybx2 and Papolb to mediate its role in spermatogenesis. We confirmed significant reduction in YBX2 and PAPOLB protein levels in testis tissue from Bnc1+/tr and Bnc1tr/tr males compared with wild-type mice (Bnc1+/+). Consistently, knockdown of Bnc1 led to downregulation of Ybx2 and Papolb in CRL-2196 cells in vitro. To investigate if BNC1 directly induces Ybx2 and Papolb gene expression, chromatin immunoprecipitation using mouse testicular tissue and luciferase reporter assays in HEK293 cells were used to identify functional binding of BNC1 to the Ybx2 and Papolb promoters at defined BNC1 binding sites. Taken together, this study reveals a mechanism for BNC1\'s role in spermatogenesis by directly binding to BNC1 binding elements in the promoter regions of both Ybx2 and Papolb and inducing transcription of these important spermatogenesis genes.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a type of oral malignancy. Long non-coding RNAs (lncRNAs) have been shown to be related to the occurrence and development of many cancers. Here, we aimed to study the role and molecular mechanism of lncRNA Homeobox A11 antisense RNA (HOXA11-AS) in OSCC.
METHODS: The expression levels of HOXA11-AS, miR-98-5p and Y box binding protein 2 (YBX2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8), flow cytometry and transwell assays were utilized to determine the proliferation, apoptosis, migration and invasion of OSCC cells. Western blot (WB) analysis was conducted to measure the levels of apoptosis, epithelial-mesenchymal transition (EMT), proliferation-related proteins and YBX2 protein. Besides, Dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull down assays were carried out to examine the relationship among HOXA11-AS, miR-98-5p and YBX2. The mice xenograft models were constructed to further determine the effect of HOXA11-AS on OSCC tumor growth in vivo.
RESULTS: HOXA11-AS was highly expressed in OSCC tissues and cells. Knockdown of HOXA11-AS significantly reduced proliferation, migration, invasion and EMT, while promoted apoptosis of OSCC cells. MiR-98-5p was a target of HOXA11-AS, and its inhibitor could revert the inhibition effect of silenced-HOXA11-AS on the progression of OSCC. Also, YBX2 was a target of miR-98-5p, and its overexpression could invert the suppression effect of miR-98-5p overexpression on the progression of OSCC. YBX2 expression was regulated by HOXA11-AS and miR-98-5p. Furthermore, HOXA11-AS silencing could reduce the tumor growth of OSCC in vivo.
CONCLUSIONS: HOXA11-AS plays an active role in the progression of OSCC, and the discovery of HOXA11-AS/miR-98-5p/YBX2 axis provides new therapeutic targets for OSCC.

Oral squamous cell carcinoma (OSCC), the subtype of head and neck cancers, is notorious for its high incidence and death rate. The role of long non-coding RNAs (lncRNAs) is discovered to be significant for the canceration and cancer progression. Long intergenic non-protein coding RNA 958 (LINC00958) is discovered as a carcinogene in multiple cancers, such as gastric cancer, pancreatic cancer, and glioma, but there has been no report about how LINC00958 functions in OSCC. The objective of our study is to unfold function and mechanism investigation on LINC00958 in OSCC. First, TCGA database showed the upregulation and prognostic significance of LINC00958 in head and neck squamous carcinoma. Then, we discovered in OSCC clinical samples that LINC00958 presented high expression and predicted poor prognosis. Also, LINC00958 was elevated in OSCC cells. In vitro gain- and loss-function experiments proved that LINC00958 facilitated cell growth, retarded apoptosis, accelerated migration, and epithelial-to-mesenchymal transition (EMT) in OSCC. Mechanistically, we confirmed the cytoplasmic expression of LINC00958 in OSCC cells, and revealed that LINC00958 sequestered miR-627-5p to upregulate YBX2 expression. Rescue assays indicated that LINC00958 regulated OSCC cell proliferation, motility and EMT through YBX2. Together, we showed that LINC00958 promoted OSCC progression through miR-627-5p/YBX2 axis, indicating LINC00958 as a new prognostic marker, and provided new perspectives for molecular targeted treatment for OSCC.

The expression of many genes during the postmeiotic stages of spermatogenesis is largely regulated by germ cell-specific RNA-binding proteins at the level of posttranscription. One of these RNA-binding proteins, YBX2, participates in mRNA storage and regulation of translation in haploid spermatids. How YBX2-stored mRNAs become translationally competent during spermiogenesis remains unknown. In the present study, we report for the first time that YBX2 interacts with PAIP1, a protein translation enhancer, in vitro and in vivo. In murine testes, PAIP1 is highly expressed and colocalizes with YBX2 in round spermatids. Using sequential RNA immunoprecipitation and sequence analysis, we identified a group of spermiogenic mRNAs indirectly bound by PAIP1 through YBX2. Translation of mRNAs bearing the YBX2 target sequence was significantly blocked by YBX2 protein, but was reinitiated when YBX2 was coexpressed with PAIP1 in vitro. Taken together, these results indicate that PAIP1 regulates the translation of YBX2-masked mRNAs during spermiogenesis, and we propose this YBX2-PAIP1 interaction plays an important role in male germ cell development.

OBJECTIVE: The etiology of infertility is still unknown in almost half of all male infertility patients. In sperm, DNA condensation differs from somatic and female gamete cells, with the protamine (PRM) gene and its transcription factor, Y-box binding protein 2 (YBX2), playing key roles in making the structure more compact. Protamine polymorphisms have been studied in different populations, but various results have been acquired.
METHODS: In our study, we examined, for the first time in a Turkish population, the association between protamine gene alleles (PRM1 c.-190C>A, PRM1 c.197G>T, and PRM2 c.248C>T), and YBX2 (c.187T>C and c.1095 + 16A>G) and male infertility. This was accomplished using polymerase chain reaction-restriction fragment length polymorphism analyses of 100 infertile and 100 fertile Turkish men. Sperm DNA fragmentation analysis was performed using the Comet technique.
RESULTS: We found that the AA and CA genotypes of the PRM1 c.-190C>A polymorphism had a significant association with infertility (p < 0.001) and the AA genotype was also highly significantly associated with high sperm DNA damage (p < 0.001).
CONCLUSIONS: This study demonstrates that the PRM1 c.-190C>A polymorphism is associated with sperm DNA fragmentation, which may impact male infertility in the Turkish population. Further research with larger groups and in various other study populations will be required to clarify the impact of protamine and YBX2 gene polymorphisms on male infertility.

BACKGROUND: Although aberrant protamine (PRM) ratios have been observed in infertile men, the mechanisms that implicit the uncoupling of PRM1 and PRM2 expression remain unclear. To uncover these mechanisms, in this observational study we have compared the PRM1/PRM2 mRNA ratio and mRNA contents of two regulatory factors of these genes.
METHODS: In this experimental study, sampling was performed by a multi-step method from 50 non-obstructive azoospermic and 12 normal men. After RNA extraction and cDNA synthesis, real-time quantitative polymerase chain reaction (RT- QPCR) was used to analyze the PRM1, PRM2, Y box binding protein 2 (YBX2) and JmjC-containing histone demethylase 2a (JHDM2A) genes in testicular biopsies of the studied samples.
RESULTS: The PRM1/PRM2 mRNA ratio differed significantly among studied groups, namely 0.21 ± 0.13 in azoospermic samples and -0.8 ± 0.22 in fertile samples. The amount of PRM2 mRNA, significantly reduced in azoospermic patients. Azoospermic men exhibited significant under expression of YBX2 gene compared to controls (P<0.001). mRNA content of this gene showed a positive correlation with PRM mRNA ratio (R=0.6, P=0.007). JHDM2A gene expression ratio did not show any significant difference between the studied groups (P=0.3). We also observed no correlation between JHDM2A mRNA content and the PRM mRNA ratio (R=0.2, P=0.3).
CONCLUSIONS: We found significant correlation between the aberrant PRM ratio (PRM2 under expression) and lower YBX2 mRNA content in testicular biopsies of azoospermic men compared to controls, which suggested that downregulation of the YBX2 gene might be involved in PRM2 under expression. These molecules could be useful biomarkers for predicting male infertility.