scholarly journals The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development

2019 ◽  
Vol 29 (4) ◽  
pp. 591-604 ◽  
Author(s):  
Soma Dash ◽  
Lindy K Brastrom ◽  
Shaili D Patel ◽  
C Anthony Scott ◽  
Diane C Slusarski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Eye Development ◽  
Ocular Tissue ◽  
Binding Motif ◽  
Mobility Shift ◽  
Developmental Defects ◽  
Protein Levels

Abstract Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans—yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3’UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3’UTR. Further, we demonstrate that Sox2 3’UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24–Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

scholarly journals Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product

2015 ◽  
Vol 36 (4) ◽  
pp. 628-644 ◽  
Author(s):  
Katherine A. Braun ◽  
Kenneth M. Dombek ◽  
Elton T. Young
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Mrna Decay ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Content Type ◽  
Amp Activated Protein Kinase ◽  
Yeast Metabolic Cycle ◽  
Metabolic Cycle

In the yeastSaccharomyces cerevisiae, the switch from respiratory metabolism to fermentation causes rapid decay of transcripts encoding proteins uniquely required for aerobic metabolism. Snf1, the yeast ortholog of AMP-activated protein kinase, has been implicated in this process because inhibiting Snf1 mimics the addition of glucose. In this study, we show that theSNF1-dependentADH2promoter, or just the major transcription factor binding site, is sufficient to confer glucose-induced mRNA decay upon heterologous transcripts.SNF1-independent expression from theADH2promoter prevented glucose-induced mRNA decay without altering the start site of transcription.SNF1-dependent transcripts are enriched for the binding motif of the RNA binding protein Vts1, an important mediator of mRNA decay and mRNA repression whose expression is correlated with decreased abundance ofSNF1-dependent transcripts during the yeast metabolic cycle. However, deletion ofVTS1did not slow the rate of glucose-induced mRNA decay.ADH2mRNA rapidly dissociated from polysomes after glucose repletion, and sequences bound by RNA binding proteins were enriched in the transcripts from repressed cells. Inhibiting the protein kinase A pathway did not affect glucose-induced decay ofADH2mRNA. Our results suggest that Snf1 may influence mRNA stability by altering the recruitment activity of the transcription factor Adr1.

scholarly journals Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yogeeshwar Ajjugal ◽  
Narendar Kolimi ◽  
Thenmalarchelvi Rathinavelan
Keyword(s):  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Repeat Expansion ◽  
Mobility Shift ◽  
Cgg Repeat ◽  
Dna And Rna ◽  
Structural Choice ◽  
Sense Strand

AbstractCGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

The Vitamin D Receptor Represses Transcription of the Pituitary Transcription Factor Pit-1 Gene without Involvement of the Retinoid X Receptor

2006 ◽  
Vol 20 (4) ◽  
pp. 735-748 ◽  
Author(s):  
Samuel Seoane ◽  
Roman Perez-Fernandez
Keyword(s):  
Vitamin D ◽  
Transcription Factor ◽  
Vitamin D Receptor ◽  
Retinoid X Receptor ◽  
Breast Adenocarcinoma ◽  
Mobility Shift ◽  
Histone Deacetylase 1 ◽  
Protein Levels ◽  
Repressive Effect ◽  
Mcf 7

Abstract Pituitary transcription factor-1 (Pit-1) plays a key role in cell differentiation during organogenesis of the anterior pituitary, and as a transcriptional activator for the pituitary GH and prolactin genes. However, Pit-1 is also expressed in nonpituitary cell types and tissues. In breast tumors, Pit-1 mRNA and protein levels are increased with respect to normal breast, and in MCF-7 human breast adenocarcinoma cells, Pit-1 increases GH secretion and cell proliferation. We report here that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration to MCF-7 cells induces a significant decrease in Pit-1 mRNA and protein levels. By deletion analyses, we mapped a region (located between −147 and −171 bp from the transcription start site of the Pit-1 gene) that is sufficient for the repressive response to 1,25-(OH)2D3. Gel mobility shift and chromatin immunoprecipitation assays confirmed the direct interaction between the vitamin D receptor (VDR) as homodimer (without the retinoid X receptor), and the Pit-1 promoter, supporting the view that Pit-1 is a direct transcriptional target of VDR. Our data also indicate that recruitment of histone deacetylase 1 is involved in this repressive effect. This ligand-dependent Pit-1 gene inhibition by VDR in the absence of the retinoid X receptor seems to indicate a new mechanism of transcriptional repression by 1,25-(OH)2D3.

scholarly journals Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

1995 ◽  
Vol 15 (1) ◽  
pp. 476-487 ◽  
Author(s):  
X Guo ◽  
Y P Zhang ◽  
D A Mitchell ◽  
D T Denhardt ◽  
A F Chambers
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Uv Light ◽  
Metastatic Potential ◽  
Binding Motif ◽  
Binding Assays ◽  
Mobility Shift ◽  
Related Transcription Factor ◽  
Altered Gene ◽  
Alpha V Beta 3

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

scholarly journals Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

Molecular dissection of a genome defense pathway in Neurospora crassa

2015 ◽  
Author(s):  
◽  
Erin C. Boone
Keyword(s):  
Neurospora Crassa ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Periphery ◽  
Bimolecular Fluorescence Complementation ◽  
Proper Function ◽  
Rnai Pathway ◽  
Binding Motif ◽  
Perinuclear Region ◽  
A Genome

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Meiotic silencing by unpaired DNA (MSUD) is an RNA interference (RNAi) pathway in Neurospora crassa that detects genes without a homologous partner and silences them for the duration of sexual development. In this study, we have further elucidated the function of known MSUD proteins, identified novel proteins that are required for MSUD, and demonstrated the conservation of RNAi-related processes at the nuclear periphery. We began by showing SAD-2 is crucial for the localization of other MSUD proteins in the perinuclear region. These data suggest that SAD-2 works as a scaffold protein and that proper function of MSUD, like other germline RNAi-like systems, is reliant on the presence of silencing proteins in the perinuclear region. An MSUD suppression assay identified two novel MSUD proteins, SAD-Y and SAD-B'. Even though SAD-Y and its homologs contain a conserved putative RNA- binding motif, they have yet to be assigned to a biochemical pathway. Our work here has linked silencing to SAD-Y-like proteins. SAD-Y has been shown to interact with other MSUD factors in both the nucleus and at the nuclear periphery. SAD-B's homolog has been found in the nuage, an epicenter for RNA-binding proteins involved in post-transcriptional regulation for Drosophila germline cells. SAD-B interacts with core MSUD proteins and has an especially intimate association with SMS-2, which requires it for localization. Furthermore, bimolecular fluorescence complementation (BiFC) revealed that SAD-B' interacts with a Golgi retrograde transport protein and an autophagy marker protein, suggesting the importance of the endomembrane system in this RNAi process.

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3263-3274 ◽  
Author(s):  
G.M. Souza ◽  
A.M. da Silva ◽  
A. Kuspa
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mrna Levels ◽  
Wild Type ◽  
Mobility Shift ◽  
Developmental Defects ◽  
C Protein ◽  
Protein Levels ◽  
Cell Extracts ◽  
Puf Protein

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.

scholarly journals Translational induction of ATF4 during integrated stress response requires noncanonical initiation factors eIF2D and DENR

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Deepika Vasudevan ◽  
Sarah D. Neuman ◽  
Amy Yang ◽  
Lea Lough ◽  
Brian Brown ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Stress Responses ◽  
Rna Binding ◽  
Open Reading Frames ◽  
Binding Motif ◽  
Integrated Stress Response ◽  
Developmental Defects ◽  
Initiation Factors

Abstract The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5′ leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5′ leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.

scholarly journals RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice

2019 ◽  
Vol 20 (8) ◽  
pp. 1965 ◽  
Author(s):  
Cosmin Cătălin Mustăciosu ◽  
Adela Banciu ◽  
Călin Mircea Rusu ◽  
Daniel Dumitru Banciu ◽  
Diana Savu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Transcriptional Control ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Diabetic Mice

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

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