scholarly journals Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific mRNAs in Chromatoid Bodies During Spermatogenesis

2020 ◽  
Vol 8 ◽  
Author(s):  
Rajakumar Anbazhagan ◽  
Raghuveer Kavarthapu ◽  
Steven L. Coon ◽  
Maria L. Dufau
Keyword(s):  
Germ Cell ◽  
Translational Regulation ◽  
Rna Transport ◽  
Rna Seq ◽  
Rna Helicases ◽  
Differential Abundance ◽  
Dead Box ◽  
Specific Mrnas ◽  
Chromatoid Bodies

GRTH/DDX25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H) show loss of the phospho-species from cytoplasm with preservation of the non-phospho form in the cytoplasm and nucleus. GRTH KI mice are sterile and lack elongated spermatids and spermatozoa, with spermatogenic arrest at step 8 of round spermatids which contain chromatoid body (CB) markedly reduced in size. We observed an absence of phospho-GRTH in CB of GRTH KI mice. RNA-Seq analysis of mRNA isolated from CB revealed that 1,421 genes show differential abundance, of which 947 genes showed a decrease in abundance and 474 genes showed an increase in abundance in GRTH KI mice. The transcripts related to spermatid development, differentiation, and chromatin remodeling (Tnp1/2, Prm1/2/3, Spem1/2, Tssk 2/3/6, Grth, tAce, and Upf2) were reduced, and the transcripts encoding for factors involved in RNA transport, regulation, and surveillance and transcriptional and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were increased in the CB of KI mice and were further validated by qPCR. In the round spermatids of wild-type mice, mRNAs of Tnp2, Prm2, and Grth were abundantly co-localized with MVH protein in the CB, while in GRTH KI mice these were minimally present. In addition, GRTH binding to Tnp1/2, Prm1/2, Grth, and Tssk6 mRNAs was found to be markedly decreased in KI. These results demonstrate the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.

scholarly journals Targeted knock-in mice with a human mutation in GRTH/DDX25 reveals the essential role of phosphorylated GRTH in spermatid development during spermatogenesis

2019 ◽  
Vol 28 (15) ◽  
pp. 2561-2572 ◽  
Author(s):  
Raghuveer Kavarthapu ◽  
Rajakumar Anbazhagan ◽  
Murugananthkumar Raju ◽  
Chon-Hwa Tsai Morris ◽  
James Pickel ◽  
...  
Keyword(s):  
Essential Role ◽  
Round Spermatid ◽  
Dependent Manner ◽  
Testis Size ◽  
Rna Helicases ◽  
Dead Box ◽  
Chromatoid Bodies ◽  
Human Mutation ◽  
Related Proteins

Abstract Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis specific member of the DEAD-box family of RNA helicases expressed in meiotic and haploid germ cells which plays an essential role in spermatogenesis. There are two species of GRTH the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in azoospermic men that when expressed in COS1-cells lack the phospho-form of GRTH. To investigate the role of the phospho-GRTH species in spermatogenesis, we generated a GRTH knock-in (KI) transgenic mice with the R242H mutation. GRTH-KI mice are sterile with reduced testis size, lack sperm with spermatogenic arrest at round spermatid stage and loss of the cytoplasmic phospho-GRTH species. Electron microscopy studies revealed reduction in the size of chromatoid bodies (CB) of round spermatids (RS) and germ cell apoptosis. We observed absence of phospho-GRTH in the CB of RS. Complete loss of chromatin remodeling and related proteins such as TP2, PRM2, TSSK6 and marked reduction of their respective mRNAs and half-lives were observed in GRTH-KI mice. We showed that phospho-GRTH has a role in TP2 translation and revealed its occurrence in a 3′ UTR dependent manner. These findings demonstrate the relevance of phospho-GRTH in the structure of the chromatoid body, spermatid development and completion of spermatogenesis and provide an avenue for the development of a male contraceptive.

scholarly journals Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

2013 ◽  
Vol 42 (5) ◽  
pp. 3298-3313 ◽  
Author(s):  
Anastasiia Kamenska ◽  
Wei-Ting Lu ◽  
Dorota Kubacka ◽  
Helen Broomhead ◽  
Nicola Minshall ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Translation ◽  
Developmental Model ◽  
Model Systems ◽  
P Bodies ◽  
Specific Mrnas

Abstract A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

scholarly journals Long non-coding RNAs (lncRNAs) in spermatogenesis and male infertility

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Meghali Joshi ◽  
Singh Rajender
Keyword(s):  
Male Infertility ◽  
Germ Cell ◽  
Germ Cells ◽  
Motile Sperm ◽  
Rna Seq ◽  
Small Noncoding Rnas ◽  
Cellular Processes ◽  
Human Male ◽  
Non Coding Rnas

Abstract Background Long non-coding RNAs (lncRNAs) have a size of more than 200 bp and are known to regulate a host of crucial cellular processes like proliferation, differentiation and apoptosis by regulating gene expression. While small noncoding RNAs (ncRNAs) such as miRNAs, siRNAs, Piwi-interacting RNAs have been extensively studied in male germ cell development, the role of lncRNAs in spermatogenesis remains largely unknown. Objective In this article, we have reviewed the biology and role of lncRNAs in spermatogenesis along with the tools available for data analysis. Results and conclusions Till date, three microarray and four RNA-seq studies have been undertaken to identify lncRNAs in mouse testes or germ cells. These studies were done on pre-natal, post-natal, adult testis, and different germ cells to identify lncRNAs regulating spermatogenesis. In case of humans, five RNA-seq studies on different germ cell populations, including two on sperm, were undertaken. We compared three studies on human germ cells to identify common lncRNAs and found 15 lncRNAs (LINC00635, LINC00521, LINC00174, LINC00654, LINC00710, LINC00226, LINC00326, LINC00494, LINC00535, LINC00616, LINC00662, LINC00668, LINC00467, LINC00608, and LINC00658) to show consistent differential expression across these studies. Some of the targets of these lncRNAs included CENPB, FAM98B, GOLGA6 family, RPGR, TPM2, GNB5, KCNQ10T1, TAZ, LIN28A, CDKN2B, CDKN2A, CDKN1A, CDKN1B, CDKN1C, EZH2, SUZ12, VEGFA genes. A lone study on human male infertility identified 9879 differentially expressed lncRNAs with three (lnc32058, lnc09522, and lnc98497) of them showing specific and high expression in immotile sperm in comparison to normal motile sperm. A few lncRNAs (Mrhl, Drm, Spga-lncRNAs, NLC1-C, HongrES2, Tsx, LncRNA-tcam1, Tug1, Tesra, AK015322, Gm2044, and LncRNA033862) have been functionally validated for their roles in spermatogenesis. Apart from rodents and humans, studies on sheep and bull have also identified lncRNAs potentially important for spermatogenesis. A number of these non-coding RNAs are strong candidates for further research on their roles in spermatogenesis.

scholarly journals Emerging Role of Eukaryote Ribosomes in Translational Control

2019 ◽  
Vol 20 (5) ◽  
pp. 1226 ◽  
Author(s):  
Nicole Dalla Venezia ◽  
Anne Vincent ◽  
Virginie Marcel ◽  
Frédéric Catez ◽  
Jean-Jacques Diaz
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
Translational Efficiency ◽  
Regulate Gene Expression ◽  
Translational Activity ◽  
Technological Advances ◽  
The Status ◽  
Specific Mrnas ◽  
Different Populations

Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of “specialized ribosomes”, which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.

scholarly journals OR02-03 Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific MRNAs in Chromatoid Bodies During Spermiogenesis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Rajakumar Anbazhagan ◽  
Raghuveer Kavarthapu ◽  
Maria L Dufau
Keyword(s):  
Translational Regulation ◽  
Rna Helicase ◽  
Cytoplasmic Organelle ◽  
Rna Helicases ◽  
Chromatin Remodelers ◽  
Chromatin Compaction ◽  
Expression Of Genes ◽  
Chromatoid Bodies ◽  
Composition Structure

Abstract Gonadotropin-regulated testicular RNA helicase (GRTH/DDX 25) is a member of the DEAD-box family of RNA helicases which play an essential role in spermatogenesis. There are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in the Japanese azoospermic men which resulted in the lack of phospho-GRTH (pGRTH) in in vitro studies. GRTH knock-in (KI) mice with insertion of the human mutant GRTH gene show loss of the cytoplasmic 61 KDa phospho-species with preservation of the non-phospho nuclear form. KI mice are sterile, lack elongated spermatids and spermatozoa with arrest at step 8 of round spermatids (RS) which contain chromatoid bodies (CB) markedly reduced in size. CB is a non-membranous, cytoplasmic organelle present adjacent to the nucleus of RS, where mRNAs bound to GRTH transported from nucleus to cytoplasmic sites are temporarily stored, translationally repressed for later transport to polyribosomes for translation at specific stages of spermiogenesis. Owing to the specific function of CBs and importance of pGRTH in spermatid elongation, CBs isolated from germ cells of WT and GRTH KI mice were used for subsequent experiments. CBs isolated from GRTH KI mice are smaller, highly condensed and lack the nuage texture of CBs in WT mice. We observed the absence of pGRTH in CB of round spermatids of GRTH KI mice. Also, MVH protein (recognized CB marker protein) was decreased in the CB of GRTH KI mice. Expression of genes related to spermatid regulation, chromatin compaction, remodeling (TP1 and 2, PRM1 and 2, GRTH, TSSK6, HMG2, GCNF, RNF8, TDRD 1, 6, 7 and 9) analyzed by qPCR were markedly reduced in the CB of GRTH KI mice compared to WT. No change was observed in the expression of bromodomain mRNAs and protein, indicating that pGRTH does not participate in the translational regulation of this protein class at the level of this organelle. Notably, mRNAs of TP2, PRM2 and GRTH which associated with GRTH protein were co-localized with MVH protein in the CB. This indicated the relevance of GRTH as a binder/transport protein of key chromatin remodelers for ensuring their mRNA repression/stability within the CB. In addition, GRTH binding to genes essential for spermatid development and regulation (TP1 and 2, PRM1 and 2, GRTH, TSSK6, RNF8 and GCNF) were also found to be markedly decreased in the CB KI mice. These results demonstrate the importance of pGRTH in the maintenance of biochemical composition/structure of the CB and role in spermatid regulation, chromatin compaction, spermatid development and completion of spermatogenesis.

scholarly journals Host cellular RNA helicases regulate SARS-CoV-2 infection

2021 ◽  
Author(s):  
Yasuo Ariumi
Keyword(s):  
Rna Helicase ◽  
Viral Rna ◽  
Rna Helicases ◽  
Granule Formation ◽  
Multifunctional Protein ◽  
Body Formation ◽  
N Protein ◽  
Dead Box ◽  
Rna Genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largest RNA genome of approximately 30kb among RNA viruses. The DDX DEAD-box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. As well, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to utilize own viral replication and overcomes their anti-viral effect of DDX21 and MOV10 through as interaction with host cellular RNA helicase. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection.

scholarly journals Orb prevents autophagy in the Drosophila germline through translational repression of Atg12 mRNA

2014 ◽  
Author(s):  
Isabelle Busseau ◽  
Stephanie Pierson ◽  
Dany Severac ◽  
Christelle Dantec ◽  
Martine Simonelig
Keyword(s):  
Translational Regulation ◽  
Molecular Level ◽  
Translational Repression ◽  
Specific Gene ◽  
Environmental Cues ◽  
Mrna Levels ◽  
Major Mode ◽  
Specific Mrnas ◽  
Essential Function

Drosophila Orb, the homologue of vertebrate CPEB is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb has also an essential function during early oogenesis which has not been addressed at the molecular level. Here, we show that orb prevents cell death during early stages of oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy, by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. The uncontrolled autophagy observed in orb mutant ovaries is reduced when Atg12 mRNA levels are decreased. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.

scholarly journals DDX23 is a Metabolic Regulator in Hepatocellular Carcinoma

2020 ◽  
Author(s):  
Jianlong Zhou ◽  
Xiaoming Wang ◽  
Jing Liang ◽  
Chaohui Tan ◽  
Changnan Chen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Rna Sequencing ◽  
High Expression ◽  
Rna Seq ◽  
Loss Of Function ◽  
Rna Helicases ◽  
Poor Survival ◽  
Hcc Cells

Abstract Background: Although biochemical activities of RNA helicases have been well-studied, physiological meaning of those factors in both normal and disease condition remained to be clarified.Methods: RNA sequencing (RNA-seq) in HCC cells indicated DDX23 are highly expressed in HCC and high expression of DDX23 is responsible for poor survival of HCC patients. Next, The expression of DDX23 was establish for subsequent investigation. The roll of DDX23 in HCC was identified by RNA-seq, RT-qPCR, LC-MS, OCR, ECAR. The effect of DDX23 on proliferative, Cloning information as well as tumorigenicity of transfected cells in mice was examined using loss-of-function experiments.Results: Here, we investigated a new role of RNA helicase in a member of the DEAD box protein family, DDX23 in hepatocellular carcinoma (HCC). RNA level of DDX23 are highly expressed in HCC and high expression of DDX23 is responsible for poor survival of HCC patients. In addition, we demonstrated that DDX23 expression is important for in vitro and in vivo tumorigenesis. RNA sequencing (RNA-seq) in HCC cells indicated that metabolism is the most affected pathway by the DDX23 and most abundant DDX23-interacting RNA are involved in metabolism in HCC, especially glycolysis. Conclusions: These findings provide new insights on the unexpected HCC-related role of DDX23, an opportunities for the development of the therapeutic target which is a master regulator of genes involved in HCC-favorable metabolic reprogram at the RNA level.

scholarly journals The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

2021 ◽  
Author(s):  
Praewa Suthapot ◽  
Tiaojiang Xiao ◽  
Gary Felsenfeld ◽  
Suradej Hongeng ◽  
Patompon Wongtrakoongate
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Differentiation ◽  
Rna Helicases ◽  
Dead Box ◽  
Rna Remodeling ◽  
Genome Wide ◽  
Mature Neurons ◽  
Stem Cell State

Understanding human neurogenesis is critical toward regenerative medicine for neurodegeneration. However, little is known how neural differentiation is regulated by RNA helicases, which comprise a diverse class of RNA remodeling enzymes. We show here that expression of the DEAD box-containing RNA helicases DDX5 and DDX17 is abundant throughout retinoic acid-induced neural differentiation of the human pluripotent stem cell (hPSC) line NTERA2, and is mostly localized within the nucleus. Using ChIP-seq, we identify that the two RNA helicases occupy chromatin genome-wide at regions associated with neurogenesis- and differentiation-related genes in both hPSCs and their neural derivatives. Further, RNA-seq analyses indicate both DDX5 and DDX17 are mutually required for controlling transcriptional expression of these genes. We show that the two RNA helicases are not important for maintenance of stem cell state of hPSCs. In contrast, they facilitate early neural differentiation of hPSCs, generation of neurospheres from the stem cells, and expression of key neurogenic transcription factors during neural differentiation. Importantly, DDX5 and DDX17 are important for differentiation of hPSCs toward NESTIN- and TUBB3-positive cells, which represent neural progenitors and mature neurons. Collectively, our findings suggest the role of DDX5 and DDX17 in transcriptional regulation of genes involved in neurogenesis, and hence in neural differentiation of hPSCs.

scholarly journals The CCR4–NOT Deadenylase Complex Maintains Adipocyte Identity

2019 ◽  
Vol 20 (21) ◽  
pp. 5274 ◽  
Author(s):  
Akinori Takahashi ◽  
Shohei Takaoka ◽  
Shungo Kobori ◽  
Tomokazu Yamaguchi ◽  
Sara Ferwati ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Gene Expression Analysis ◽  
Rna Seq ◽  
Normal Body Temperature ◽  
Metabolic Genes ◽  
Brown Adipose ◽  
Specific Mrnas ◽  
Tissue Characteristics ◽  
And Function

Shortening of poly(A) tails triggers mRNA degradation; hence, mRNA deadenylation regulates many biological events. In the present study, we generated mice lacking the Cnot1 gene, which encodes an essential scaffold subunit of the CCR4–NOT deadenylase complex in adipose tissues (Cnot1-AKO mice) and we examined the role of CCR4–NOT in adipocyte function. Cnot1-AKO mice showed reduced masses of white adipose tissue (WAT) and brown adipose tissue (BAT), indicating abnormal organization and function of those tissues. Indeed, Cnot1-AKO mice showed hyperinsulinemia, hyperglycemia, insulin resistance, and glucose intolerance and they could not maintain a normal body temperature during cold exposure. Muscle-like fibrous material appeared in both WAT and BAT of Cnot1-AKO mice, suggesting the acquisition of non-adipose tissue characteristics. Gene expression analysis using RNA-sequencing (RNA-seq) showed that the levels of adipose tissue-related mRNAs, including those of metabolic genes, decreased, whereas the levels of inflammatory response-related mRNAs increased. These data suggest that the CCR4–NOT complex ensures proper adipose tissue function by maintaining adipocyte-specific mRNAs at appropriate levels and by simultaneously suppressing mRNAs that would impair adipocyte function if overexpressed.

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