scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yarden Katz ◽  
Feifei Li ◽  
Nicole J Lambert ◽  
Ethan S Sokol ◽  
Wai-Leong Tam ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
State Regulation ◽  
Ribosome Profiling ◽  
Luminal Cell ◽  
Mesenchymal Transition ◽  
Cell State

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

scholarly journals TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

2020 ◽  
Author(s):  
Hua Jin ◽  
Daxiang Na ◽  
Reazur Rahman ◽  
Weijin Xu ◽  
Allegra Fieldsend ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Growth Control ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Mtor Inhibition ◽  
Mrna Targets ◽  
Specific Mrnas ◽  
Specific Mrna

Abstract4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

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 CLIP-Seq and massively parallel functional analysis of the CELF6 RNA binding protein reveals a role in destabilizing synaptic gene mRNAs through interaction with 3’UTR elements in vivo

2018 ◽  
Author(s):  
Michael A. Rieger ◽  
Dana M. King ◽  
Barak A. Cohen ◽  
Joseph D. Dougherty
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Synaptic Proteins ◽  
Massively Parallel ◽  
Sequence Motifs ◽  
Mrna Targets ◽  
The Brain

AbstractCELF6 is a RNA-binding protein in a family of proteins with roles in human health and disease, however little is known about the mRNA targets or in vivo function of this protein. We utilized CLIP-Seq to identify, for the first time, in vivo targets of CELF6 and identify hundreds of transcripts bound by CELF6 in the brain. We found these are disproportionately mRNAs coding for synaptic proteins. We then conducted functional validation of these targets, testing greater than 400 CELF6 bound sequence elements for their activity, applying a massively parallel reporter assay framework to evaluation of the CLIP data. We also mutated potential binding motifs within these elements and tested their impact. This comprehensive analysis led us to ascribe a previously unknown function to CELF6: we found bound elements were generally repressive of translation, that CELF6 further enhances this repression via decreasing RNA abundance, and this process was dependent on UGU-rich sequence motifs. This greatly extends the known role for CELF6, which had previously been defined only as a splicing factor. We further extend these findings by demonstrating the same function for CELF3, CELF4, and CELF5. Finally, we demonstrate that the CELF6 targets are derepressed in CELF6 mutant mice in vivo, confirming this new role in the brain. Thus, our study demonstrates that CELF6 and other sub-family members are repressive CNS RNA-binding proteins, and CELF6 downregulates specific mRNAs in vivo.

scholarly journals Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

2018 ◽  
Author(s):  
Yuhki Saito ◽  
Yuan Yuan ◽  
Ilana Zucker-Scharff ◽  
John J. Fak ◽  
Yoko Tajima ◽  
...  
Keyword(s):  
Motor Coordination ◽  
Rna Binding ◽  
Synapse Formation ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
Knock Out ◽  
Knock Out Mice ◽  
Cerebellar Purkinje Cells ◽  
The Brain

SUMMARYRNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

scholarly journals The RNA binding protein FXR1 is a new driver in the 3q26-29 amplicon and predicts poor prognosis in human cancers

2015 ◽  
Vol 112 (11) ◽  
pp. 3469-3474 ◽  
Author(s):  
Jun Qian ◽  
Mohamed Hassanein ◽  
Megan D. Hoeksema ◽  
Bradford K. Harris ◽  
Yong Zou ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Lung Squamous Cell Carcinoma ◽  
Regulatory Function ◽  
Driver Gene ◽  
Aberrant Expression ◽  
Human Cancers

Aberrant expression of RNA-binding proteins has profound implications for cellular physiology and the pathogenesis of human diseases such as cancer. We previously identified the Fragile X-Related 1 gene (FXR1) as one amplified candidate driver gene at 3q26-29 in lung squamous cell carcinoma (SCC). FXR1 is an autosomal paralog of Fragile X mental retardation 1 and has not been directly linked to human cancers. Here we demonstrate that FXR1 is a key regulator of tumor progression and its overexpression is critical for nonsmall cell lung cancer (NSCLC) cell growth in vitro and in vivo. We identified the mechanisms by which FXR1 executes its regulatory function by forming a novel complex with two other oncogenes, protein kinase C, iota and epithelial cell transforming 2, located in the same amplicon via distinct binding mechanisms. FXR1 expression is a candidate biomarker predictive of poor survival in multiple solid tumors including NSCLCs. Because FXR1 is overexpressed and associated with poor clinical outcomes in multiple cancers, these results have implications for other solid malignancies.

Molecular Functions of the Mammalian Fragile X Mental Retardation Protein: Insights Into Mental Retardation and Synaptic Plasticity

2013 ◽  
Author(s):  
Claudia Bagni ◽  
Eric Klann
Keyword(s):  
Mental Retardation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Rna Binding Protein ◽  
Fragile X Mental Retardation ◽  
The Family ◽  
Mental Retardation Protein ◽  
The Brain

Chapter 8 discusses how Fragile X syndrome (FXS) is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP is highly expressed in the brain and gonads, the two organs mainly affected in patients with the syndrome. Functionally, FMRP belongs to the family of RNA-binding proteins, shuttling from the nucleus to the cytoplasm, and, as shown for other RNA-binding proteins, forms large messenger ribonucleoparticles.

scholarly journals TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

2020 ◽  
Vol 6 (33) ◽  
pp. eabb8771 ◽  
Author(s):  
Hua Jin ◽  
Weijin Xu ◽  
Reazur Rahman ◽  
Daxiang Na ◽  
Allegra Fieldsend ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Growth Control ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Mtor Inhibition ◽  
Mrna Targets ◽  
Specific Mrnas ◽  
Specific Mrna

4E-BP (eIF4E-BP) represses translation initiation by binding to the 5′ cap–binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

FMRP association with and regulation of Fragile X granules exhibit circuit-dependent requirements for the KH2 RNA binding domain

2018 ◽  
Author(s):  
Ellen C. Gingrich ◽  
Katherine A. Shepard ◽  
Molly E. Mitchell ◽  
Kirsty Sawicka ◽  
Jennifer C. Darnell ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Brain Regions ◽  
Binding Domain ◽  
Cell Type ◽  
Related Disorder ◽  
Rna Granules ◽  
Rna Binding Domain

AbstractThe localization and translation of mRNAs is controlled by a diverse array of ribonucleoprotein particles (RNPs), multimolecular complexes containing mRNAs and RNA binding proteins. Fragile X granules (FXGs) are a family of RNPs that exemplify the diversity of RNA granules in the mammalian nervous system. FXGs are found in a conserved subset of neurons, where they localize exclusively to the axonal compartment. Notably, the specific RNA binding proteins and mRNAs found in FXGs depend on brain circuit and neuron type, with all forebrain FXGs containing Fragile X mental retardation protein (FMRP), the protein mutated in the human autism-related disorder Fragile X syndrome. FMRP negatively regulates FXG abundance but is not required for their association with ribosomes or mRNA. To better understand the circuit-dependent mechanisms whereby FMRP associates with and regulates FXGs, we asked how a disease-causing point mutation, I304N, in the KH2 RNA binding domain of FMRP affects these granules in two brain regions – cortex and hippocampus. We found that FMRPI304N had a reduced association with FXGs, as it was absent from approximately half of FXGs in cortex and nearly all FXGs in hippocampus. FXG abundance correlated with the number of FMRP-containing FXGs, suggesting that FMRP regulates FXG abundance by KH2-independent mechanisms that occur locally within the granules. Together, these findings illustrate that cell type-dependent mechanisms guide the assembly of similar RNA granules. Further, point mutations in RNA granule components may lead to cell type-dependent phenotypes that produce atypical forms of disorders that normally arise from more severe mutations.

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