Structure and dynamics of the quaternary hunchback mRNA translation repression complex

ABSTRACTA key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

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Surveying the global landscape of post-transcriptional regulators

10.1101/2021.08.09.455688 ◽

2021 ◽

Author(s):

Kendra Reynaud ◽

Anna M McGeachy ◽

David Noble ◽

Zuriah A Meacham ◽

Nicholas Ingolia

Keyword(s):

Rna Binding ◽

Mrna Translation ◽

Transcriptional Regulators ◽

Regulatory Activity ◽

Modular Architecture ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Binding Domains ◽

Fluorescence Measurements ◽

Transcriptional Gene Regulation

Numerous proteins regulate gene expression by modulating mRNA translation and decay. In order to uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.

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Faculty Opinions recommendation of Comprehensive Identification of RNA-Binding Domains in Human Cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726579306.793531390 ◽

2017 ◽

Author(s):

Julie Forman-Kay ◽

Andrew Chong

Keyword(s):

Rna Binding ◽

Human Cells ◽

Binding Domains ◽

Rna Binding Domains

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RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014894 ◽

2020 ◽

pp. jbc.RA120.014894

Author(s):

Ravi Kumar ◽

Dipak Kumar Poria ◽

Partho Sarothi Ray

Keyword(s):

Inflammatory Response ◽

Chronic Inflammation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Suppressor Gene ◽

Cooperative Action ◽

Translation Repression

Post-transcriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a pro-inflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) HuR in response to LPS stimulation, but the role of other regulatory factors remain unknown. Here we report that the RBP Lupus antigen (La) interacts with the 3’UTR of PDCD4 mRNA and prevents miR-21-mediated translation repression. While LPS causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

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RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽

10.3390/v13030361 ◽

2021 ◽

Vol 13 (3) ◽

pp. 361

Author(s):

Rui-Zhu Shi ◽

Yuan-Qing Pan ◽

Li Xing

Keyword(s):

Virus Replication ◽

Rna Binding ◽

Rna Viruses ◽

Rna Helicase ◽

Rna Helicase A ◽

Double Stranded Rna ◽

Binding Domains ◽

N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

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In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽

10.1093/genetics/159.4.1617 ◽

2001 ◽

Vol 159 (4) ◽

pp. 1617-1630

Author(s):

Suk-Won Jin ◽

Nancy Arno ◽

Adam Cohen ◽

Amy Shah ◽

Qijin Xu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cell ◽

Rna Binding ◽

Dominant Negative ◽

Missense Mutations ◽

Biochemical Tests ◽

Cell Fates ◽

Cytoplasmic Polyadenylation ◽

Binding Domains ◽

Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

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The Role of Translation Initiation Regulation in Haematopoiesis

Comparative and Functional Genomics ◽

10.1155/2012/576540 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Godfrey Grech ◽

Marieke von Lindern

Keyword(s):

Gene Expression ◽

Translation Initiation ◽

Translational Control ◽

Molecular Mechanisms ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Translation Control ◽

Haematological Disorders

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

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Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

Biochemical Journal ◽

10.1042/bj20050694 ◽

2005 ◽

Vol 393 (1) ◽

pp. 245-254 ◽

Cited By ~ 32

Author(s):

Catherine Martel ◽

Paolo Macchi ◽

Luc Furic ◽

Michael A. Kiebler ◽

Luc Desgroseillers

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Binding Activity ◽

Nuclear Trafficking ◽

Binding Domains ◽

Bipartite Nuclear Localization Signal ◽

Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

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The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽

10.1242/dev.126.13.2841 ◽

1999 ◽

Vol 126 (13) ◽

pp. 2841-2853 ◽

Cited By ~ 1

Author(s):

G. Deshpande ◽

G. Calhoun ◽

P.D. Schedl

Keyword(s):

Fusion Protein ◽

Rna Binding ◽

Chimeric Protein ◽

Dominant Negative ◽

Terminal Domain ◽

Binding Domains ◽

Transcriptional Regulatory ◽

Dominant Negative Activity ◽

Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

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