Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5′ and 3′ untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex.

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Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Nucleic Acids Research ◽

10.1093/nar/gkaa144 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4507-4520 ◽

Cited By ~ 6

Author(s):

Smriti Pandey ◽

Chandra M Gravel ◽

Oliver M Stockert ◽

Clara D Wang ◽

Courtney L Hegner ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Site Directed Mutagenesis ◽

Genetic Identification ◽

Messenger Rnas ◽

Functional Surface

Abstract The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5′ and 3′ untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ’s interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ’s NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

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The RNA-Binding Protein CsrA Controls Virulence in Erwinia amylovora by Regulating RelA, RcsB, and FlhD at the Posttranscriptional Level

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-19-0077-r ◽

2019 ◽

Vol 32 (10) ◽

pp. 1448-1459 ◽

Cited By ~ 5

Author(s):

Jae Hoon Lee ◽

Veronica Ancona ◽

Tiyakhon Chatnaparat ◽

Ho-wen Yang ◽

Youfu Zhao

Keyword(s):

Molecular Mechanisms ◽

Erwinia Amylovora ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Direct Interaction ◽

Antagonistic Effect ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Posttranscriptional Level

CsrA, an RNA-binding protein, binds to target transcripts and alters their translation or stability. In Erwinia amylovora, CsrA positively regulates the expression of type III secretion system (T3SS), exopolysaccharide amylovoran, and motility. In this study, the global effect of CsrA and its noncoding small RNA (ncsRNA) csrB in E. amylovora was determined by RNA-seq, and potential molecular mechanisms of CsrA-dependent virulence regulation were examined. Transcriptomic analyses under the T3SS-inducing condition revealed that mutation in the csrA gene led to differential expression of more than 20% of genes in the genome. Among them, T3SS genes and those required for cell growth and viability were significantly downregulated. On the other hand, the csrB mutant exhibited significant upregulation of most major virulence genes, suggesting an antagonistic effect of csrB on CsrA targets. Direct interaction between CsrA protein and csrB was further confirmed through the RNA electrophoretic mobility shift assay (REMSA). However, no direct interaction between CsrA and hrpL and hrpS transcripts was detected, suggesting that HrpL and HrpS are not targets of CsrA, whereas three CsrA targets (relA, rcsB, and flhD) were identified and confirmed by REMSA, site-directed mutagenesis, and LacZ reporter gene assays. These findings might partially explain how CsrA positively controls E. amylovora virulence by targeting major regulators at the posttranscriptional level.

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Genetic Analysis of Functional Domains Within the Drosophila LARK RNA-Binding Protein

Genetics ◽

10.1093/genetics/159.1.229 ◽

2001 ◽

Vol 159 (1) ◽

pp. 229-240

Author(s):

Gerard P McNeil ◽

Andrew J Schroeder ◽

Mary A Roberts ◽

F Rob Jackson

Keyword(s):

Genetic Analysis ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Site Directed Mutagenesis ◽

Female Sterility ◽

Circadian Regulation ◽

Rna Recognition Motif ◽

Structural Domains

Abstract LARK is an essential Drosophila RNA-binding protein of the RNA recognition motif (RRM) class that functions during embryonic development and for the circadian regulation of adult eclosion. LARK protein contains three consensus RNA-binding domains: two RRM domains and a retroviral-type zinc finger (RTZF). To show that these three structural domains are required for function, we performed a site-directed mutagenesis of the protein. The analysis of various mutations, in vivo, indicates that the RRM domains and the RTZF are required for wild-type LARK functions. RRM1 and RRM2 are essential for viability, although interestingly either domain can suffice for this function. Remarkably, mutation of either RRM2 or the RTZF results in the same spectrum of phenotypes: mutants exhibit reduced viability, abnormal wing and mechanosensory bristle morphology, female sterility, and flightlessness. The severity of these phenotypes is similar in single mutants and double RRM2; RTZF mutants, indicating a lack of additivity for the mutations and suggesting that RRM2 and the RTZF act together, in vivo, to determine LARK function. Finally, we show that mutations in RRM1, RRM2, or the RTZF do not affect the circadian regulation of eclosion, and we discuss possible interpretations of these results. This genetic analysis demonstrates that each of the LARK structural domains functions in vivo and indicates a pleiotropic requirement for both the LARK RRM2 and RTZF domains.

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RNA–Binding Protein HuD as a Versatile Factor in Neuronal and Non–Neuronal Systems

Biology ◽

10.3390/biology10050361 ◽

2021 ◽

Vol 10 (5) ◽

pp. 361

Author(s):

Myeongwoo Jung ◽

Eun-Kyung Lee

Keyword(s):

Gene Expression ◽

Neural Plasticity ◽

Molecular Mechanisms ◽

Neuronal Development ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Novel Treatments ◽

Target Mrnas ◽

Neuronal Systems

HuD (also known as ELAVL4) is an RNA–binding protein belonging to the human antigen (Hu) family that regulates stability, translation, splicing, and adenylation of target mRNAs. Unlike ubiquitously distributed HuR, HuD is only expressed in certain types of tissues, mainly in neuronal systems. Numerous studies have shown that HuD plays essential roles in neuronal development, differentiation, neurogenesis, dendritic maturation, neural plasticity, and synaptic transmission by regulating the metabolism of target mRNAs. However, growing evidence suggests that HuD also functions as a pivotal regulator of gene expression in non–neuronal systems and its malfunction is implicated in disease pathogenesis. Comprehensive knowledge of HuD expression, abundance, molecular targets, and regulatory mechanisms will broaden our understanding of its role as a versatile regulator of gene expression, thus enabling novel treatments for diseases with aberrant HuD expression. This review focuses on recent advances investigating the emerging role of HuD, its molecular mechanisms of target gene regulation, and its disease relevance in both neuronal and non–neuronal systems.

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The RNA-binding protein ELAVL1/HuR is essential for mouse spermatogenesis, acting both at meiotic and postmeiotic stages

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0212 ◽

2011 ◽

Vol 22 (16) ◽

pp. 2875-2885 ◽

Cited By ~ 39

Author(s):

Mai Nguyen Chi ◽

Jacques Auriol ◽

Bernard Jégou ◽

Dimitris L. Kontoyiannis ◽

James M.A. Turner ◽

...

Keyword(s):

Germ Cells ◽

Posttranscriptional Regulation ◽

Target Gene ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Female Sterility ◽

Targeted Deletion

Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accurate protein synthesis during germ cell development relies on RNA binding proteins that control the storage, stability, and translation of mRNAs in a tightly and temporally regulated manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regulation in somatic cells but the function of which during gametogenesis has never been investigated. In this study, we have used conditional loss- and gain-of-function approaches to address this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads to male but not female sterility. Mutant males are azoospermic because of the extensive death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter defect is also observed upon HuR overexpression. To elucidate further the molecular mechanisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2, a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifically binds hspa2 mRNA and controls its expression at the translational level in germ cells. Our study provides the first genetic evidence of HuR involvement during spermatogenesis and reveals Hspa2 as a target for HuR.

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RNA-BINDING PROTEIN HUR BLOCKADE HAS POTENT ANTI-TUMOR ACTIVITIES IN HUMAN RENAL CELL CARCINOMA BOTH IN VITRO AND IN VIVO

The Journal of Urology ◽

10.1016/s0022-5347(09)60440-7 ◽

2009 ◽

Vol 181 (4S) ◽

pp. 153-153 ◽

Cited By ~ 1

Author(s):

Sabrina Danilin ◽

Lionel Thomas ◽

Thomas Charles ◽

Carole Sourbier ◽

Véronique Lindner ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Human Renal Cell Carcinoma

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The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo

PLoS ONE ◽

10.1371/journal.pone.0134751 ◽

2015 ◽

Vol 10 (9) ◽

pp. e0134751 ◽

Cited By ~ 8

Author(s):

John A. Gaynes ◽

Hideo Otsuna ◽

Douglas S. Campbell ◽

John P. Manfredi ◽

Edward M. Levine ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Axon Outgrowth

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The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3194 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3194-3201 ◽

Cited By ~ 157

Author(s):

R J Buckanovich ◽

R B Darnell

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Motor Dysfunction ◽

High Affinity ◽

Loop Region ◽

Nuclear Rna ◽

Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

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The RNA-Binding Protein HuR Promotes Cell Migration and Cell Invasion by Stabilizing the β-actin mRNA in a U-Rich-Element-Dependent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.00113-07 ◽

2007 ◽

Vol 27 (15) ◽

pp. 5365-5380 ◽

Cited By ~ 79

Author(s):

Virginie Dormoy-Raclet ◽

Isabelle Ménard ◽

Eveline Clair ◽

Ghada Kurban ◽

Rachid Mazroui ◽

...

Keyword(s):

Hela Cells ◽

Molecular Mechanisms ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Migration And Invasion ◽

Actin Stress Fiber ◽

Dependent Manner ◽

Mrna Stabilization ◽

Actin Protein

ABSTRACT A high expression level of the β-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the β-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the β-actin mRNA by associating with a uridine-rich element within its 3′ untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the β-actin mRNA. HuR depletion in HeLa cells alters key β-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated β-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation.

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