Connecting cis-elements and trans-factors with mechanisms of developmental regulation of mRNA translation in meiotic and haploid mammalian spermatogenic cells

mRNA-specific regulation of translational activity plays major roles in directing the development of meiotic and haploid spermatogenic cells in mammals. Although many RNA-binding proteins (RBPs) have been implicated in normal translational control and sperm development, little is known about the keystone of the mechanisms: the interactions of RBPs and microRNAs withcis-elements in mRNA targets. The problems in connecting factors and elements with translational control originate in the enormous complexity of post-transcriptional regulation in mammalian cells. This creates confusion as to whether factors have direct or indirect and large or small effects on the translation of specific mRNAs. This review argues that gene knockouts, heterologous systems, and overexpression of factors cannot provide convincing answers to these questions. As a result, the mechanisms involving well-studied mRNAs (Ddx4/Mvh,Prm1,Prm2, andSycp3) and factors (DICER1, CPEB1, DAZL, DDX4/MVH, DDX25/GRTH, translin, and ELAV1/HuR) are incompletely understood. By comparison, mutations in elements can be used to define the importance of specific pathways in regulating individual mRNAs. However, few elements have been studied, because the only reliable system to analyze mutations in elements, transgenic mice, is considered impractical. This review describes advances that may facilitate identification of the direct targets of RBPs and analysis of mutations incis-elements. The importance of upstream reading frames in the developmental regulation of mRNA translation in spermatogenic cells is also documented.

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Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

10.1101/2021.11.19.469243 ◽

2021 ◽

Author(s):

Omer Ziv ◽

Svetlana Farberov ◽

Jian You Lau ◽

Eric A Miska ◽

Grzegorz Kudla ◽

...

Keyword(s):

Rna Structure ◽

Mammalian Cells ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Features ◽

Structural Domain ◽

Mrna Targets ◽

Structural Principles ◽

Pumilio Proteins

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

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CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

10.1101/2021.07.08.451585 ◽

2021 ◽

Author(s):

Mickaële Hemono ◽

Alexandre Haller ◽

Johana Chicher ◽

Anne-Marie Duchêne ◽

Richard Patryk Ngondo

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mitochondrial Protein ◽

Embryonic Stem ◽

Nuclear Genome ◽

Mrna Translation ◽

Mitochondrial Proteins ◽

Molecular Function ◽

First Time

Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

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Pat1 proteins: regulating mRNAs from birth to death?

BioMolecular Concepts ◽

10.1515/bmc-2012-0005 ◽

2012 ◽

Vol 3 (4) ◽

pp. 295-306

Author(s):

Nancy Standart ◽

Aline Marnef

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Mrna Decay ◽

Rna Binding ◽

Rna Binding Proteins ◽

Viral Gene Expression ◽

Model Systems ◽

Viral Gene ◽

Nuclear Speckles ◽

Mrna Targets

AbstractThe Pat1 protein family has been the subject of several recent extensive investigations of diverse model systems ranging from yeast, flies and worms to man, using a variety of experimental approaches. Although some contradictions remain, the emerging consensus view is that these RNA-binding proteins act in mRNA decay by physically linking deadenylation with decapping and by regulating gene expression as translational repressors. These multiple functions are present in the single invertebrate Pat1 proteins, whereas, in vertebrates, one Pat1 variant represses translation in early development, while a somatic version synthesised in embrogenesis and in adults acts in mRNA decay. At steady state, Pat1 proteins are found enriched in cytoplasmic P(rocessing)-bodies, and related mRNP complexes and granules. Evidence recently obtained from mammalian tissue culture cells shows that Pat1 shuttles in and out of the nucleus, where it localises to nuclear speckles, PML bodies and nucleolar caps, which suggests RNA-related nuclear functions. Less well understood, Pat1 proteins may play additional roles in miRNA silencing and/or biogenesis, as well in the regulation of viral gene expression. Due to the relatively low level of sequence conservation between Pat1 proteins from different species and lacking any discernable motifs, determining their functional domains has proved difficult, as is obtaining a simple unified view of the location of the binding sites of their interacting proteins in all examined species. Questions that remain to be addressed include the following: 1) What are their roles in the nucleus? 2) What is the link, if one exists, between their cytoplasmic and nuclear roles? 3) Do they have specific mRNA targets? 4) Which signalling pathways regulate their P-body localisation in mammalian cells, which may affect quiescent cell survival?

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The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins

BMC Biology ◽

10.1186/s12915-021-01213-y ◽

2022 ◽

Vol 20 (1) ◽

Author(s):

Mickaële Hémono ◽

Alexandre Haller ◽

Johana Chicher ◽

Anne-Marie Duchêne ◽

Richard Patryk Ngondo

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Nuclear Genome ◽

Mrna Translation ◽

Mitochondrial Proteins ◽

Molecular Function ◽

First Time ◽

Tpr Domain

Abstract Background Mitochondria require thousands of proteins to fulfill their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability, or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Results Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal stable RNA-independent interactions of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. We show that this interaction occurs during the process of active translation and is dependent on CLUH TPR domain. Conclusions Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by revealing new partners and by highlighting its link to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

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LARP6C regulates selective mRNA translation to promote pollen tube guidance in Arabidopsis thaliana

10.1101/2020.11.27.401307 ◽

2020 ◽

Author(s):

Elodie Billey ◽

Said Hafidh ◽

Isabel Cruz-Gallardo ◽

Celso G. Litholdo ◽

Viviane Jean ◽

...

Keyword(s):

Pollen Tube ◽

Rna Binding ◽

Cell Function ◽

Rna Binding Proteins ◽

Binding Protein ◽

Mrna Translation ◽

Guided Growth ◽

Pollen Tube Guidance ◽

Paracrine Signalling ◽

Mrna Targets

ABSTRACTIn angiosperms, non-motile sperm cells are delivered to the ovules for fertilization via a guided growth of the pollen tube. RNA binding proteins are key regulators that control mRNA fate post-transcriptionally and thus essential for normal cell function. But very little is known on the mechanistic bases of mRNA regulations and trans-acting factors governing male-female signalling. Here we demonstrate that the evolutionarily conserved RNA binding protein LARP6C is necessary for pollen tube guidance and fertilization. larp6c loss-of-function mutants exhibit male induced fertility defects as mutant pollen tubes frequently are unable to find ovules for fertilization. In mature pollen, LARP6C localises with a pollen specific poly(A) binding protein to cytoplasmic foci likely containing mRNPs. With RNA immunoprecipitation and sequencing, we demonstrate that LARP6C is associated in vivo with mRNAs required for pollen tube guidance or polarized cell growth. We further demonstrate using in vitro and in planta transient assays that LARP6C binds 5’-UTR box motifs to orchestrate the balance between translation, decay and storage of its mRNA targets. We propose a model where LARP6C maintains its mRNA target in translationally silent state likely to promote localized translation and guided pollen tube growth upon paracrine signalling.

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RNA-binding protein IGF2BP2/IMP2 is required for laminin-β2 mRNA translation and is modulated by glucose concentration

AJP Renal Physiology ◽

10.1152/ajprenal.00185.2012 ◽

2012 ◽

Vol 303 (1) ◽

pp. F75-F82 ◽

Cited By ~ 20

Author(s):

Valerie Schaeffer ◽

Kim M. Hansen ◽

David R. Morris ◽

Renée C. LeBoeuf ◽

Christine K. Abrass

Keyword(s):

Diabetic Nephropathy ◽

Actin Cytoskeleton ◽

Translational Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Mrna Translation ◽

Diabetic Rats ◽

Laminin Β2

Laminin-β2 (LAMB2) is a critical component of the glomerular basement membrane as content of LAMB2 in part determines glomerular barrier permeability. Previously, we reported that high concentrations of glucose reduce expression of this laminin subunit at the translational level. The present studies were undertaken to further define systems that control Lamb2 translation and the effect of high glucose on those systems. Complementary studies were performed using in vitro differentiation of cultured podocytes and mesangial cells exposed to normal and elevated concentrations of glucose, and tissues from control and diabetic rats. Together, these studies provide evidence for regulation of Lamb2 translation by IMP2, an RNA binding protein that targets Lamb2 mRNA to the actin cytoskeleton. Expression of Imp2 itself is regulated by the transcription factor HMGA2, which in turn is regulated by the microRNA let-7b. Elevated concentrations of glucose increase let-7b, which reduces HMGA2 expression, in turn reducing IMP2 and LAMB2. Correlative changes in kidney tissues from control and streptozotocin-induced diabetic rats suggest these control mechanisms are operative in vivo and may contribute to proteinuria in diabetic nephropathy. To our knowledge, this is the first time that translation of Lamb2 mRNA has been linked to the actin cytoskeleton, as well as to specific RNA-binding proteins. These translational control points may provide new targets for therapy in proteinuric disorders such as diabetic nephropathy where LAMB2 levels are reduced.

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Signalling to translation: how signal transduction pathways control the protein synthetic machinery

Biochemical Journal ◽

10.1042/bj20070024 ◽

2007 ◽

Vol 403 (2) ◽

pp. 217-234 ◽

Cited By ~ 350

Author(s):

Christopher G. Proud

Keyword(s):

Protein Synthesis ◽

Protein Kinases ◽

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Signalling Pathways ◽

Cancer Tissue ◽

Energy Status ◽

Specific Mrnas

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.

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Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e13-08-0495 ◽

2014 ◽

Vol 25 (21) ◽

pp. 3413-3423 ◽

Cited By ~ 12

Author(s):

David Dávila ◽

Eva M. Jiménez-Mateos ◽

Claire M. Mooney ◽

Guillermo Velasco ◽

David C. Henshall ◽

...

Keyword(s):

Oxidative Stress ◽

Neuronal Death ◽

Rna Binding ◽

Rna Binding Proteins ◽

Gene Activation ◽

Mrna Translation ◽

Granule Neurons ◽

Cellular Mechanisms ◽

Hsp27 Expression ◽

Specific Regulation

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

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Multi-modal regulation of C. elegans hermaphrodite spermatogenesis by the GLD-1-FOG-2 complex

10.1101/386250 ◽

2018 ◽

Author(s):

Shuang Hu ◽

Lauren E. Ryan ◽

Ebru Kaymak ◽

Lindsay Freeberg ◽

Te-Wen Lo ◽

...

Keyword(s):

Sex Determination ◽

Germ Cell ◽

Germ Cells ◽

Translational Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Wild Type ◽

C Elegans ◽

Target Mrnas ◽

Mrna Targets

AbstractProper germ cell sex determination in Caenorhabditis nematodes requires a network of RNA-binding proteins (RBPs) and their target mRNAs. In some species, changes in this network enabled limited XX spermatogenesis, and thus self-fertility. In C. elegans, one of these selfing species, the global sex-determining gene tra-2 is regulated in germ cells by a conserved RBP, GLD-1, via the 3’ untranslated region (3’UTR) of its transcript. A C. elegans-specific GLD-1 cofactor, FOG-2, is also required for hermaphrodite sperm fate, but how it modifies GLD-1 function is unknown. Germline feminization in gld-1 and fog-2 null mutants has been interpreted as due to cell-autonomous elevation of TRA-2 translation. Consistent with the proposed role of FOG-2 in translational control, the abundance of nearly all GLD-1 target mRNAs (including tra-2) is unchanged in fog-2 mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss of gld-1 function elevates germline TRA-2 expression to detectable levels, but loss of fog-2 function does not. A simple quantitative model of tra-2 activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly, fog-2 and gld-1 activity enable the sperm fate even when GLD-1 cannot bind to the tra-2 3’ UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites within tra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as “hyperrepressors” of tra-2 activity. These variants bind RNA more weakly in vitro than does wild-type GLD-1. These results indicate that gld-1 and fog-2 regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in the C. elegans hermaphrodite is far from complete.

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The DAZL and PABP families: RNA-binding proteins with interrelated roles in translational control in oocytes

Reproduction ◽

10.1530/rep-08-0524 ◽

2009 ◽

Vol 137 (4) ◽

pp. 595-617 ◽

Cited By ~ 70

Author(s):

Matthew Brook ◽

Joel W S Smith ◽

Nicola K Gray

Keyword(s):

Gene Expression ◽

Germ Cells ◽

Translational Control ◽

Translational Regulation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor

Gametogenesis is a highly complex process that requires the exquisite temporal, spatial and amplitudinal regulation of gene expression at multiple levels. Translational regulation is important in a wide variety of cell types but may be even more prevalent in germ cells, where periods of transcriptional quiescence necessitate the use of post-transcriptional mechanisms to effect changes in gene expression. Consistent with this, studies in multiple animal models have revealed an essential role for mRNA translation in the establishment and maintenance of reproductive competence. While studies in humans are less advanced, emerging evidence suggests that translational regulation plays a similarly important role in human germ cells and fertility. This review highlights specific mechanisms of translational regulation that play critical roles in oogenesis by activating subsets of mRNAs. These mRNAs are activated in a strictly determined temporal manner via elements located within their 3′UTR, which serve as binding sites fortrans-acting factors. While we concentrate on oogenesis, these regulatory events also play important roles during spermatogenesis. In particular, we focus on the deleted in azoospermia-like (DAZL) family of proteins, recently implicated in the translational control of specific mRNAs in germ cells; their relationship with the general translation initiation factor poly(A)-binding protein (PABP) and the process of cytoplasmic mRNA polyadenylation.

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