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Combinatorial mRNA Regulation by miRNAs and Pumilio proteins [PUM1CLIP]

ABSTRACT: Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3′ untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3′ UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3′ UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent.

ORGANISM(S): Homo sapiens

PROVIDER: GSE110519 | GEO | 2018/10/17

REPOSITORIES: GEO

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Combinatorial mRNA Regulation by miRNAs and Pumilio proteins [CLIP-seq]

Project description:Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3′ untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3′ UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3′ UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent.

2018-10-17 | GSE110518 | GEO

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [PAR-CLIP]

Project description:Tristetraprolin/ZFP36/TTP and ELAVL1/HuR are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embyonic kidney cells and examined combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Among the targets ZFP36 binds and negatively regulates the mRNA of genes encoding proteins necessary for immune function and cancer, and other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance from ZFP36 overexpression independent of effects from confounding features, such as 3M-bM-^@M-^Y UTR length. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes. ZFP36 preferentially interacts with and regulates AU-rich sequences while ELAVL1 prefers predominantly U- and CU-rich sequences. RNA target specificity identified by global in vivo ZFP36-mRNA interactions were quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 both bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to untangle the in vivo combinatorial regulation by RNA-binding proteins. FLAG-HA ZFP36

2013-12-24 | E-GEOD-53184 | biostudies-arrayexpress

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [Affymetrix]

2013-12-24 | E-GEOD-53183 | biostudies-arrayexpress

Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [HuR]

Project description:The 3’ UTR of messenger RNAs serves as the regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins (RBPs). Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3’ UTR sites have emerged in recent studies. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RBP, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3’UTRs and assess their co-occurrence. Additionally, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2-HuR overlap, 13 candidates were screened in luciferase reporter assays, compared to miRNA site mutant controls. Eleven of the sites showed a repressive activity, which displayed significant de-repression upon subsequent testing of the reporters in Dicer-null cells, substantiating miRNA dependence. To experimentally test for HuR’s role in co-regulation, we tested the reporters in CRISPR-generated HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA-HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3’ UTR.

2018-01-19 | GSE102320 | GEO

Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [Ago2]

2018-01-19 | GSE102319 | GEO

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [PAR-CLIP]

Project description:Tristetraprolin/ZFP36/TTP and ELAVL1/HuR are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embyonic kidney cells and examined combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Among the targets ZFP36 binds and negatively regulates the mRNA of genes encoding proteins necessary for immune function and cancer, and other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance from ZFP36 overexpression independent of effects from confounding features, such as 3’ UTR length. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes. ZFP36 preferentially interacts with and regulates AU-rich sequences while ELAVL1 prefers predominantly U- and CU-rich sequences. RNA target specificity identified by global in vivo ZFP36-mRNA interactions were quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 both bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to untangle the in vivo combinatorial regulation by RNA-binding proteins.

2013-12-24 | GSE53184 | GEO

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [Affymetrix]

Project description:Tristetraprolin/ZFP36/TTP and ELAVL1/HuR are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embyonic kidney cells and examined combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Among the targets ZFP36 binds and negatively regulates the mRNA of genes encoding proteins necessary for immune function and cancer, and other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance from ZFP36 overexpression independent of effects from confounding features, such as 3’ UTR length. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes. ZFP36 preferentially interacts with and regulates AU-rich sequences while ELAVL1 prefers predominantly U- and CU-rich sequences. RNA target specificity identified by global in vivo ZFP36-mRNA interactions were quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 both bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to untangle the in vivo combinatorial regulation by RNA-binding proteins.

2013-12-24 | GSE53183 | GEO

Pumilio regulates mRNA decay during the late maternal-to-zygotic transition in Drosophila melanogaster

Project description:Pumilio (PUM) is a Drosophila member of a conserved family of sequence-specific RNA-binding proteins that have been shown to regulate mRNA stability and/or translation in a variety of organisms. PUM has been shown to repress the translation of several mRNAs in the Drosophila early embryo; failure to repress these targets leads to lethal developmental defects. Here we use a combination of microarray-based gene expression profiling and next-generation sequencing to identify more than 200 mRNAs that are associated with full-length PUM protein in early embryos and to define a global role for PUM in mRNA decay. Surprisingly, despite the fact that PUM is maternally supplied and thus is present from the beginning of embryogenesis, the vast majority of PUM-directed decay occurs only after zygotic genome activation. We show that the smaug mRNA, which itself encodes an RNA-binding protein that directs transcript decay, is a direct target of PUM via binding sites in the smg 3'UTR. Whereas the endogenous smaug mRNA and the transgenic reporter mRNA that carries the smaug 3'UTR undergo decay after zygotic genome activation, a reporter with an array of PUM-binding sites decays before zygotic genome activation. These data support a model in which additional cis-elements in the smg 3'UTR delay decay until after zygotic genome activation.

2017-07-01 | GSE48645 | GEO

NORAD-induced phase separation of PUMILIO proteins is required for genome maintenance

Project description:Long noncoding RNAs (lncRNAs) can regulate the activity of interacting RNA binding proteins (RBPs), but how lncRNAs efficiently compete against other transcripts to bind and regulate RBP activity is poorly understood. This problem is exemplified by NORAD, a lncRNA that maintains genomic stability by inhibiting PUMILIO (PUM) proteins. Here we show that NORAD is able to out-compete other PUM-binding transcripts by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to sequester a super-stoichiometric amount of PUM in these condensates. Accordingly, synthetic RNAs that induce PUM phase separation rescue genome instability in NORAD-deficient cells. These results illuminate mechanisms of RBP regulation by RNA-driven phase separation and uncover an essential role for this process in genome maintenance.

2021-03-25 | GSE154812 | GEO

Global Profiling of RNA-Binding Protein Target Sites by LACE-seq

Project description:RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of an RBP in those rare cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of cDNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4, and PTBP1, in mature oocytes. Unexpectedly, transcriptomic and proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes could fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP binding sites in a few cells opens the door for studying the roles of RBPs in embryonic development and reproductive diseases.

2021-04-25 | GSE137925 | GEO

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