Translational control in germ cell development: A role for the RNA-binding proteins Musashi-1 and Musashi-2

In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.

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The Dynamic Regulation of mRNA Translation and Ribosome Biogenesis During Germ Cell Development and Reproductive Aging

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710186 ◽

2021 ◽

Vol 9 ◽

Author(s):

Marianne Mercer ◽

Seoyeon Jang ◽

Chunyang Ni ◽

Michael Buszczak

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Ribosome Biogenesis ◽

Binding Proteins ◽

Rna Binding ◽

Mrna Translation ◽

Cell Development ◽

Reproductive Aging ◽

Germ Cell Development ◽

And Function

The regulation of mRNA translation, both globally and at the level of individual transcripts, plays a central role in the development and function of germ cells across species. Genetic studies using flies, worms, zebrafish and mice have highlighted the importance of specific RNA binding proteins in driving various aspects of germ cell formation and function. Many of these mRNA binding proteins, including Pumilio, Nanos, Vasa and Dazl have been conserved through evolution, specifically mark germ cells, and carry out similar functions across species. These proteins typically influence mRNA translation by binding to specific elements within the 3′ untranslated region (UTR) of target messages. Emerging evidence indicates that the global regulation of mRNA translation also plays an important role in germ cell development. For example, ribosome biogenesis is often regulated in a stage specific manner during gametogenesis. Moreover, oocytes need to produce and store a sufficient number of ribosomes to support the development of the early embryo until the initiation of zygotic transcription. Accumulating evidence indicates that disruption of mRNA translation regulatory mechanisms likely contributes to infertility and reproductive aging in humans. These findings highlight the importance of gaining further insights into the mechanisms that control mRNA translation within germ cells. Future work in this area will likely have important impacts beyond germ cell biology.

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RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development

Molecular and Cellular Biology ◽

10.1128/mcb.00676-15 ◽

2015 ◽

Vol 35 (23) ◽

pp. 4030-4042 ◽

Cited By ~ 24

Author(s):

Leah L. Zagore ◽

Sarah E. Grabinski ◽

Thomas J. Sweet ◽

Molly M. Hannigan ◽

R. Michael Sramkoski ◽

...

Keyword(s):

Alternative Splicing ◽

Germ Cell ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cell Loss ◽

Cell Development ◽

Splicing Regulation ◽

Germ Cell Development ◽

Splicing Regulator ◽

Alternatively Spliced

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.

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hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

10.21203/rs.3.rs-1060705/v1 ◽

2021 ◽

Author(s):

Shuiqiao Yuan ◽

Shenglei Feng ◽

Jinmei Li ◽

Hui Wen ◽

Kuan Liu ◽

...

Keyword(s):

Alternative Splicing ◽

Germ Cell ◽

Germ Cells ◽

Rna Binding ◽

Cell Development ◽

Mrna Splicing ◽

Conditional Knockout ◽

Cell Junction ◽

Germ Cell Development ◽

Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

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Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development

Journal of Cell Science ◽

10.1242/jcs.111.9.1255 ◽

1998 ◽

Vol 111 (9) ◽

pp. 1255-1265 ◽

Cited By ~ 1

Author(s):

D.J. Elliott ◽

K. Oghene ◽

G. Makarov ◽

O. Makarova ◽

T.B. Hargreave ◽

...

Keyword(s):

Germ Cell ◽

Cell Biology ◽

Rna Binding ◽

Spatial Association ◽

Cell Types ◽

Cell Development ◽

Mrna Splicing ◽

Human Testis ◽

Germ Cell Development ◽

Nuclear Regions

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

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Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses

International Journal of Molecular Sciences ◽

10.3390/ijms21124548 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4548 ◽

Cited By ~ 1

Author(s):

Kwanuk Lee ◽

Hunseung Kang

Keyword(s):

Abiotic Stress ◽

Plant Growth ◽

Stress Responses ◽

Translational Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Metabolism ◽

Functional Roles ◽

Growth Development

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

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Translational Control of Cytochrome c by RNA-Binding Proteins TIA-1 and HuR

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.3295-3307.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 3295-3307 ◽

Cited By ~ 136

Author(s):

Tomoko Kawai ◽

Ashish Lal ◽

Xiaoling Yang ◽

Stefanie Galban ◽

Krystyna Mazan-Mamczarz ◽

...

Keyword(s):

Er Stress ◽

Translational Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Patterns ◽

Mrna Levels ◽

Coding Region ◽

Translation Rate ◽

Translational Repressor

ABSTRACT Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stress-triggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its ∼900-bp-long, adenine- and uridine-rich 3′ untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3′UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

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Abstract P-22: Enhanced Crosslinking and Immunoprecipitation (Eclip) Data Reveal Interactions of RNA Binding Proteins with the Human Ribosome

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p22 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S21-S21

Author(s):

Andrey Buyan ◽

Ivan Kulakovskiy ◽

Sergey Dmitriev

Keyword(s):

Translational Control ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Repetitive Sequences ◽

Structural Data

Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.

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