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 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.

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 β-Actin mRNA interactome mapping by proximity biotinylation

2019 ◽  
Vol 116 (26) ◽  
pp. 12863-12872 ◽  
Author(s):  
Joyita Mukherjee ◽  
Orit Hermesh ◽  
Carolina Eliscovich ◽  
Nicolas Nalpas ◽  
Mirita Franz-Wachtel ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Embryonic Fibroblasts ◽  
Dynamic View ◽  
Kh Domain ◽  
Actin Mrna ◽  
Interactome Mapping ◽  
Specific Mrna

The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3′ UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional β-actin–associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/MARTA2 has been shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3′ UTR of β-actin mRNA and is essential for β-actin mRNA localization, but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

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 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 RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael W Webster ◽  
James AW Stowell ◽  
Lori A Passmore
Keyword(s):  
Mrna Stability ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Dissociation Rate ◽  
Sequence Motifs ◽  
Similar Sequence ◽  
Mrna Targets

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) interact with Ccr4-Not via multiple regions within low-complexity sequences, suggestive of a multipartite interface that extends beyond previously defined interactions. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. Analysis of binding kinetics reveals that this is primarily due to differences in dissociation rate constants. Consequently, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo and can be used for the prediction of mRNA targets.

scholarly journals The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins

2018 ◽  
Author(s):  
Michael W Webster ◽  
James A W Stowell ◽  
Lori A Passmore
Keyword(s):  
Mrna Stability ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Dissociation Rate ◽  
Multiple Regions ◽  
Specific Mrnas ◽  
Dissociation Rate Constants

AbstractThe Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) act as molecular tethers: They recruit Ccr4-Not via multiple regions within low-complexity sequences, and bind specific RNA sequences via RNA-binding domains. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. This is primarily due to differences in the dissociation rate constants. As a result, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo. Together, we provide new insight into the selective deadenylation of specific mRNAs by Ccr4-Not, and the prediction of targeted mRNAs.

scholarly journals Principles of mRNA targeting and regulation via the Arabidopsis m6A-binding proteins ECT2 and ECT3

2021 ◽  
Author(s):  
Laura Arribas-Hernández ◽  
Sarah Rennie ◽  
Tino Köster ◽  
Michael Schon ◽  
Carlotta Porcelli ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Super Resolution ◽  
Binding Activity ◽  
Intrinsically Disordered ◽  
Mrna Targets ◽  
Yth Domain

AbstractGene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. The Arabidopsis YTH-domain protein ECT2 is thought to influence mRNA 3’-end formation via binding to URU(m6A)Y sites, an unexpected conclusion given that ECT2 functions require its m6A binding activity, and that RR(m6A)CH is the m6A consensus site in all eukaryotes. Here, we apply the orthogonal techniques individual nucleotide-resolution UV-crosslinking and immunoprecipitation (iCLIP) and HyperTRIBE to define high-quality target sets of the YTH-domain proteins ECT2 and ECT3. The results show that in vivo, ECT2 does in fact bind to RR(m6A)CH. URUAY and other pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH islands in pyrimidine-rich regions. Such regions may also be implicated in ECT2-binding. In particular, a series of properties unique to the URUAY motif suggest that URUAY-type sequences act as sites of competition between unknown RNA-binding proteins and the intrinsically disordered region of ECT2. We also show that the abundance of many ECT2/3 mRNA targets is decreased in meristematic cells devoid of ECT2/3/4-activity. In contrast, loss of ECT2/3/4 activity has no effect on polyadenylation site usage in ECT2/3 targets, consistent with the exclusive cytoplasmic localization of ECT2 observed by super-resolution confocal microscopy. Our study reconciles conflicting results between genetic observations on N6-adenosine methylation and ECT2/3/4 function on the one side, and ECT2 target identification on the other, and point to regulation of cytoplasmic mRNA function, including abundance, as a mechanism of plant YTHDF action.

scholarly journals Ribonomic Analysis of Human Pum1 Reveals cis-trans Conservation across Species despite Evolution of Diverse mRNA Target Sets

2008 ◽  
Vol 28 (12) ◽  
pp. 4093-4103 ◽  
Author(s):  
Adam R. Morris ◽  
Neelanjan Mukherjee ◽  
Jack D. Keene
Keyword(s):  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Consensus Sequence ◽  
Puf Proteins ◽  
Mrna Targets ◽  
Puf Protein ◽  
Genes Encoding

ABSTRACT PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes.

scholarly journals Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

2021 ◽  
Vol 17 (12) ◽  
pp. e1009658
Author(s):  
Valentin Schneider-Lunitz ◽  
Jorge Ruiz-Orera ◽  
Norbert Hubner ◽  
Sebastiaan van Heesch
Keyword(s):  
Translational Regulation ◽  
Binding Proteins ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Translational Efficiency ◽  
Splicing Factors ◽  
Biological Processes ◽  
Differential Affinity

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8—all identified as multifunctional RBPs by our analysis—selectively influence target translation rates depending on 5’ UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.

Sign in / Sign up

Export Citation Format

Share Document