scholarly journals The myopathic transcription factor DUX4 induces the production of truncated RNA-binding proteins in human muscle cells

2021 ◽  
Author(s):  
Amy E Campbell ◽  
Michael C Dyle ◽  
Lorenzo Calviello ◽  
Tyler Matheny ◽  
Michael A Cortázar ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Binding ◽  
Temporal Dynamics ◽  
Rna Binding Proteins ◽  
Muscle Cells ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Potent Effect ◽  
High Concordance ◽  
Muscle Homeostasis ◽  
Human Muscle Cells

DUX4 is an embryonic transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4 dysregulates multiple pathways that could contribute to FSHD pathophysiology. However, lack of temporal data and the knowledge of which RNAs are actively translated following DUX4 expression has hindered our understanding of the cascade of events that lead to muscle cell death. Here, we interrogate the DUX4 transcriptome and translatome over time and find dysregulation of most key pathways as early as 4 hours after DUX4 induction, demonstrating the potent effect of DUX4 in disrupting muscle homeostasis. We also observe extensive transcript downregulation as well as induction, and a high concordance between mRNA abundance and translation status. Significantly, DUX4 triggers widespread production of truncated protein products derived from aberrant RNAs that are degraded in normal muscle cells. One such protein, truncated serine/arginine-rich splicing factor 3 (SRSF3-TR), is present in FSHD muscle cells and disrupts splicing autoregulation when ectopically expressed in myoblasts. Taken together, the temporal dynamics of DUX4 induction show how the pathologic presence of an embryonic transcription factor in muscle cells alters gene expression at all levels of the central dogma.

Translational regulation of ANG II type 1 receptors in proliferating vascular smooth muscle cells

2006 ◽  
Vol 290 (1) ◽  
pp. R50-R56 ◽  
Author(s):  
Sunghou Lee ◽  
Hong Ji ◽  
Zheng Wu ◽  
Wei Zheng ◽  
Ali Hassan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Muscle Cells ◽  
Distribution Analysis ◽  
Exon 2

The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT1aR) densities (Bmax) increased by 30% between 5 and 7 days in culture [Bmax (fmol/mg protein): day 5, 379 ± 8.4 vs. day 7, 481 ± 12, n = 3, P < 0.05] under conditions in which no significant changes in AT1aR mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 ± 0.01 vs. day 7, 0.24 ± 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 ± 0.14 vs. day 7, 0.33 ± 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT1aR mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT1aR mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 ± 9.9 vs. day 7, 56.8 ± 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5′ leader sequence (5′LS) of the AT1aR [5′LS RPC (AU): day 5, 0.62 ± 0.15 vs. day 7, 0.23 ± 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 ± 5.7 vs. day 7, 17.2 ± 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT1aR expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5′LS of the AT1aR mRNA.

scholarly journals Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

2019 ◽  
Author(s):  
Andreas U. Müller ◽  
Marc Leibundgut ◽  
Nenad Ban ◽  
Eilika Weber-Ban
Keyword(s):  
Dna Damage ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Information ◽  
Rna Molecules ◽  
Bacterial Transcription ◽  
Arthrobacter Aurescens

AbstractIn mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure ofArthrobacter aurescensPafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using anin vivomutational screen inMycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

scholarly journals Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice. Running title: Tcfl5 is haploinsufficient in vivo

2021 ◽  
Author(s):  
Weiya Xu ◽  
Yiyun Zhang ◽  
Dongdong Qin ◽  
Yiqian Gui ◽  
Shu Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Specific Protein ◽  
Dual Function ◽  
Specific Cell ◽  
Chip Sequencing ◽  
Dna And Rna

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

scholarly journals The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anthony J Linares ◽  
Chia-Ho Lin ◽  
Andrey Damianov ◽  
Katrina L Adams ◽  
Bennett G Novitch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Cytoskeletal Proteins ◽  
Control Programs ◽  
Neuronal Progenitor ◽  
Neuronal Genes

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

scholarly journals High-throughput intracellular biopsy of microRNAs for dissecting the temporal dynamics of cellular heterogeneity

2020 ◽  
Vol 6 (24) ◽  
pp. eaba4971
Author(s):  
Zixun Wang ◽  
Lin Qi ◽  
Yang Yang ◽  
Mingxing Lu ◽  
Kai Xie ◽  
...  
Keyword(s):  
Rna Binding ◽  
Temporal Dynamics ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Single Copy ◽  
Reaction Chamber ◽  
Cellular Heterogeneity ◽  
Cell Subpopulations ◽  
Cellular Phenotypes

The capability to analyze small RNAs responsible for post-transcriptional regulation of genes expression is essential for characterizing cellular phenotypes. Here, we describe an intracellular biopsy technique (inCell-Biopsy) for fast, multiplexed, and highly sensitive profiling of microRNAs (miRNAs). The technique uses an array of diamond nanoneedles that are functionalized with size-dependent RNA binding proteins, working as “fishing rods” to directly pull miRNAs out of cytoplasm while keeping the cells alive, thus enabling quasi-single-cell miRNA analysis. Each nanoneedle works as a reaction chamber for parallel in situ amplification, visualization, and quantification of miRNAs as low as femtomolar, which is sufficient to detect miRNAs of a single-copy intracellular abundance with specificity to single-nucleotide variation. Using inCell-Biopsy, we analyze the temporal miRNA transcriptome over the differentiation of embryonic stem cells (ESCs). The combinatorial miRNA expression patterns derived by inCell-Biopsy identify emerging cell subpopulations differentiated from ESCs and reveal the dynamic evolution of cellular heterogeneity.

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 Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development

2020 ◽  
Author(s):  
Jun Cao ◽  
Andrew L. Routh ◽  
Muge N. Kuyumcu-Martinez
Keyword(s):  
Heart Development ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Muscle Cells ◽  
Full Length ◽  
Actin Binding ◽  
Nanopore Sequencing ◽  
Mrna Isoforms

ABSTRACTAlternative splicing (AS) increases the variety of the proteome by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full length Tpm1 isoforms composed of distinct exons with specific linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. Nanopore sequencing combined with polyA sequencing revealed that the RNA binding protein RBFOX2 controls AS of muscle specific Tpm1 isoforms and expression of TPM1 protein via terminal exon splicing impacting its polyadenylation. Furthermore, RBFOX2 regulates Tpm1 AS antagonistically to PTBP1. In sum, we defined full length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of muscle specific isoforms of Tpm1 by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle enriched genes.

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 Staufen1 impairs stress granule formation in skeletal muscle cells from myotonic dystrophy type 1 patients

2016 ◽  
Vol 27 (11) ◽  
pp. 1728-1739 ◽  
Author(s):  
Aymeric Ravel-Chapuis ◽  
Amanda Klein Gunnewiek ◽  
Guy Bélanger ◽  
Tara E. Crawford Parks ◽  
Jocelyn Côté ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Muscle Cells ◽  
Cellular Stress Response ◽  
Granule Formation ◽  
A Cell ◽  
Cell Type Specific ◽  
Cug Repeats

Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUGexp) in the DMPK mRNA 3′UTR. CUGexp-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1– and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type–specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUGexp mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1.

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