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Mouse testis RNA methylation

Jiaxiu Yin, Xinxin Li, Zaili Zhang, Xiaohua Luo, Li Wang, Lin Liu
Apoptosis is a multi-step mechanism of cell self‑destruction for maintaining cellular homeostatic balance. Accumulating evidence indicates that abnormal apoptosis promotes the evolution and progression of myelodysplastic syndromes (MDS). As a novel cancer-testis antigen, sperm‑associated antigen 6 (SPAG6) has been reported to regulate apoptosis through the tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in the MDS cell line SKM‑1. However, the mechanism of the intrinsic cell death pathway for apoptosis induction by SPAG6 silencing is unclear...
May 2, 2018: International Journal of Oncology
Yusuke Sato, Daijiro Iwasawa, Kuo Ping Hui, Rena Nakagomi, Seiichi Nishizawa
Here we examined optimization of the running buffer in boronate affinity electrophoresis for improved separation of PIWI-interacting RNA (piRNA) with 2'-O-methylated ribose in 3'-terminal nucleotide. The use of Good's buffer, such as HEPES, significantly increased the separation efficiency for piRNA over normal RNA with free 3'-terminal ribose, and retained an ability to resolve the difference by at least 4-nucleotide lengths in the target piRNAs. We also demonstrated a single-step separation of piRNA from mouse testis total RNA...
2018: Analytical Sciences: the International Journal of the Japan Society for Analytical Chemistry
Ling Peng, Fengjuan Zhang, Renfu Shang, Xueyan Wang, Jiayi Chen, James J Chou, Jinbiao Ma, Ligang Wu, Ying Huang
Small noncoding RNAs (sncRNAs) regulate many genes in eukaryotic cells. Hua enhancer 1 (Hen1) is a 2'-O-methyltransferase that adds a methyl group to the 2'-OH of 3'-terminal nucleotide of sncRNAs. The types and properties of sncRNAs may vary among different species, and the domain composition, structure, and function of Hen1 proteins differ accordingly. In mammals, Hen1 specifically methylates sncRNAs called p-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs). However, other types of sncRNAs that are methylated by Hen1 have not yet been reported, and the structures and the substrates of mammalian Hen1 remain unknown...
April 27, 2018: Journal of Biological Chemistry
Jianqiang Bao, Sophie Rousseaux, Jianjun Shen, Kevin Lin, Yue Lu, Mark T Bedford
CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development...
May 18, 2018: Nucleic Acids Research
Takamasa Hirano, Hidetoshi Hasuwa, Haruhiko Siomi
The mouse testis has served as a popular model system to study a wide range of biological processes, including germ cell development, meiosis, epigenetic changes of chromatin, transposon silencing, and small RNA-mediated epigenetic modifications. PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are almost exclusively expressed in animal gonads. They repress transposons by forming effector complexes with PIWI proteins to maintain genome integrity of the germline. Here we describe detailed procedures of how to produce monoclonal antibodies against a mouse nuclear PIWI protein, MIWI2, which functions in de novo DNA methylation of target transposon loci...
2017: Methods in Molecular Biology
Kazadi Mutoji, Anukriti Singh, Thu Nguyen, Heidi Gildersleeve, Amy V Kaucher, Melissa J Oatley, Jon M Oatley, Ellen K Velte, Christopher B Geyer, Keren Cheng, John R McCarrey, Brian P Hermann
Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs...
December 2016: Biology of Reproduction
Leanne Stalker, Stewart J Russell, Carmon Co, Robert A Foster, Jonathan LaMarre
Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus...
January 2016: Biology of Reproduction
Maria B Garcia-Fabiani, Mauro A Montanaro, Ezequiel Lacunza, Elizabeth R Cattaneo, Rosalind A Coleman, Magali Pellon-Maison, Maria R Gonzalez-Baro
Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements...
October 15, 2015: Biochemical Journal
Alessandra Santillo, Sara Falvo, Paolo Chieffi, Maria Maddalena Di Fiore, Rosalba Senese, Gabriella Chieffi Baccari
D-aspartate (D-Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D-Asp indirectly stimulates spermatogenesis through the hypothalamic-pituitary-gonadal axis. Moreover, in vitro studies have demonstrated that D-Asp up-regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type-B mouse spermatogonia retaining markers of mitotic germ cells (GC-1) was employed to explore more direct involvement of D-Asp in spermatogenesis...
February 2016: Journal of Cellular Physiology
Daisuke Itou, Yusuke Shiromoto, Shin-ya Yukiho, Chika Ishii, Toru Nishimura, Narumi Ogonuki, Atsuo Ogura, Hidetoshi Hasuwa, Yoshitaka Fujihara, Satomi Kuramochi-Miyagawa, Toru Nakano
Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]...
March 30, 2015: Current Biology: CB
Amandine Vanhoutteghem, Sébastien Messiaen, Françoise Hervé, Brigitte Delhomme, Delphine Moison, Jean-Maurice Petit, Virginie Rouiller-Fabre, Gabriel Livera, Philippe Djian
Absence of mitosis and meiosis are distinguishing properties of male germ cells during late fetal and early neonatal periods. Repressors of male germ cell meiosis have been identified, but mitotic repressors are largely unknown, and no protein repressing both meiosis and mitosis is known. We demonstrate here that the zinc-finger protein BNC2 is present in male but not in female germ cells. In testis, BNC2 exists as several spliced isoforms and presumably binds to DNA. Within the male germ cell lineage, BNC2 is restricted to prospermatogonia and undifferentiated spermatogonia...
November 2014: Development
Tomoko Ichiyanagi, Kenji Ichiyanagi, Ayako Ogawa, Satomi Kuramochi-Miyagawa, Toru Nakano, Shinichiro Chuma, Hiroyuki Sasaki, Heiichiro Udono
HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28-32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24-32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α...
October 29, 2014: Nucleic Acids Research
Liuping Zhang, Jie Wang, Yaoqian Pan, Jie Jin, Jianrong Sang, Pan Huang, Genbao Shao
Histone H3 lysine 4 methylation (H3K4me) is an epigenetic modification associated with gene activation and is dynamically regulated by histone methylases and demethylases. To date, the expression patterns of H3K4me and its demethylases in the developing testis remain unclear. The present study was designed to detect the expression of H3K4me1/2/3 and its demethylases LSD1, RBP2 and SMCX in 21-, 40- and 60-day-old mouse testes by using immunohistochemistry, quantitative real-time polymerase chain reaction (PCR) and Western blot...
December 2014: Cell and Tissue Research
Misuzu Kurihara, Akira Shiraishi, Honoo Satake, Atsushi P Kimura
Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes...
August 26, 2014: Journal of Molecular Biology
Viswanath Gunda, Dennie T Frederick, Maria J Bernasconi, Jennifer A Wargo, Sareh Parangi
BACKGROUND: NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells. METHODS: We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2'-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot...
August 2014: Thyroid: Official Journal of the American Thyroid Association
Lawrence A Scheving, Xiuqi Zhang, Oscar A Garcia, Rebecca F Wang, Mary C Stevenson, David W Threadgill, William E Russell
Dsk5 mice have a gain of function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-wk-old male Dsk5 heterozygotes (+/Dsk5) were 62% heavier compared with those of wild-type controls (+/+). The livers of the +/Dsk5 mice compared with +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and nonparenchymal cells...
March 1, 2014: American Journal of Physiology. Gastrointestinal and Liver Physiology
Yunqiang Liu, Meiling Wang, Siyuan Jiang, Yongjie Lu, Dachang Tao, Yuan Yang, Yongxin Ma, Sizhong Zhang
Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression...
February 2014: BMB Reports
A Bortvin
Over the past decade, PIWI-interacting RNAs (piRNAs) have emerged as the most intriguing class of small RNAs. Almost every aspect of piRNA biology defies established rules of the RNA interference world while the scope of piRNA functional potential spans from transcriptional gene silencing to genome defense to transgenerational epigenetic phenomena. This review will focus on the genomic origins, biogenesis, and function of piRNAs in the mouse testis - an exceptionally robust experimental system amenable to genetic, cell-biological, molecular, and biochemical studies...
June 2013: Biochemistry. Biokhimii︠a︡
Yusuke Shiromoto, Satomi Kuramochi-Miyagawa, Akito Daiba, Shinichiro Chuma, Ami Katanaya, Akiko Katsumata, Ken Nishimura, Manami Ohtaka, Mahito Nakanishi, Toshinobu Nakamura, Koichi Yoshinaga, Noriko Asada, Shota Nakamura, Teruo Yasunaga, Kanako Kojima-Kita, Daisuke Itou, Tohru Kimura, Toru Nakano
piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype...
June 2013: RNA
Shobbir Hussain, Francesca Tuorto, Suraj Menon, Sandra Blanco, Claire Cox, Joana V Flores, Stephen Watt, Nobuaki R Kudo, Frank Lyko, Michaela Frye
Posttranscriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and are often organized by the chromatoid body. Here, we identify the RNA methyltransferase NSun2 as a novel component of the chromatoid body and, further, show that NSun2 is essential for germ cell differentiation in the mouse testis. In NSun2-depleted testes, genes encoding Ddx4, Miwi, and Tudor domain-containing (Tdr) proteins are repressed, indicating that RNA-processing and posttranscriptional pathways are impaired...
April 2013: Molecular and Cellular Biology
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