Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159510 | GEO | 2021/09/09
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: PUMILIO proteins are known to repress target genes by specifically binding to PUMILIO response elements (PREs) in target mRNAs. NORAD is a noncoding RNA that negatively regulates PUMILIO activity. The goal of this study was to determine the gene expression changes that result from knockout of NORAD or overexpression of PUMILIO and to test whether NORAD knockout causes PUMILIO hyperactivity. Methods: RNA-seq libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat Sample Preparation kit (Illumina) and sequenced using the 100 bp paired-end protocol on an Illumina HiSeq 2000. For comparing NORAD+/+ and NORAD-/- HCT116 cells, 3 biological replicates per genotype were sequenced. For PUM overexpression experiments, 3 replicates of GFP-expressing HCT116 cells (negative control) and 2 independent PUM1- or PUM2-overexpressing clones (2 replicates each) were sequenced. Results: Gene expression profiles show that PUMILIO target genes are downregulated in both NORAD knockout cells and PUMILIO overexpressing cells. Conclusions: These data indicate that NORAD sequesters PUMILIO, preventing excessive repression of PUMILIO target genes that are important for maintaining genomic stability.
2015-12-03 | GSE75440 | GEO
Project description:The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding site location, number, density, and context are important determinants of regulation. Moreover, the content of optimal synonymous codons exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.
2023-09-03 | GSE241940 | GEO
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 | GSE110519 | GEO
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
Project description:Pumilio proteins are RNA-binding proteins that control mRNA translation and stability by binding to the 3' UTR of target mRNAs. Mammals have two canonical Pumilio proteins, PUM1 and PUM2, which are known to act in many biological processes, including embryonic development, neurogenesis, cell cycle regulation and genomic stability. Here, we characterized a new role of both PUM1 and PUM2 in regulating cell morphology, migration, and adhesion in T-REx-293 cells, in addition to previously known defects in growth rate. Gene ontology analysis of differentially expressed genes in PUM double knockout (PDKO) cells for both cellular component and biological process showed enrichment in categories related to adhesion and migration. PDKO cells had a collective cell migration rate significantly lower than that of WT cells and displayed changes in actin morphology. In addition, during growth, PDKO cells aggregated into clusters (clumps) due to an inability to escape cell-cell contacts. Addition of extracellular matrix (Matrigel) alleviated the clumping phenotype. Collagen IV (ColIV), a major component of Matrigel, was shown to be the driving force in allowing PDKO cells to monolayer appropriately, however, ColIV protein levels remained unperturbed in PDKO cells. This study characterizes a novel cellular phenotype associated with cellular morphology, migration, and adhesion which can aid in developing better models for PUM function in both developmental processes and disease.
2023-01-01 | GSE207836 | GEO
Project description:To assess the roles of the Dcp2 C-terminal domain and the decapping activators Pat1, Lsm1, and Dhh1 in mRNA decapping, we used RNA-Seq to analyze the expression profiles of yeast cells harboring a truncation of the Dcp2 C-terminal domain, mutations that render Dcp2 catalytically inactive, or deletions of the PAT1, LSM1, and DHH1 genes. Consistent with our recent model for decapping regulation, we found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation and that loss of these control functions causes significant deregulation of mRNA decapping; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap and, as expected, are targeted to decapping-dependent decay.
2017-12-09 | GSE107841 | GEO
Project description:The human members of the PUF family of proteins, PUM1 and PUM2, are RNA-binding proteins that post-transcriptionally regulate gene expression through binding to a PUM recognition element (PRE) in the 3′ UTR of target mRNAs, promoting RNA decay. Recent RNA-seq experiments in PUM1/2 knockdown conditions have identified hundreds of known and new human PUM targets through measurement of changes in steady state RNA levels. However, steady-state RNA levels do not allow for measurement of changes in RNA stability between conditions and do not allow for the differentiation between the contributions of changes in transcription rates and changes in RNA decay. Here, we identify hundreds of human PUM1/2 targets that have changes in RNA stability following PUM1/2 knockdown. We separate the contributions of changes in transcription rate and RNA stability and find that human PUM proteins almost exclusively modulate RNA abundance through changing RNA stability and not transcription. In addition, we find that the sequence preferences for all possible 8mers are largely similar between PUM1 and PUM2, suggesting that PUM1 and PUM2 recognize similar targets. We identify an ideal PRE “rulebook” by determining key contextual features around PREs, including local AU content, location of a PRE within a 3′ UTR, clustering of PREs, and number of miRNA sites near a PRE, that help differentiate functional PREs from non-functional ones as measured by our decay dataset. Consistent with previously identified functional roles of mammalian PUMs, we find that human PUM1 and PUM2 modulate the decay of genes related to signaling cascades and neuronal function. Finally, we train machine learning models to predict functional regulation of RNA targets by the human PUM proteins and find that contextual features around PREs contribute meaningful information to our models.
2020-08-05 | GSE145237 | GEO
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
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
Project description:We used RNA-seq to measure transcript levels in human cells with the PUM1 and PUM2 proteins knocked down; by analyzing the differences in transcript abundances between those conditions, we were able to identify 927 targets showing significant changes in the presence of the PUM knockdown.
2017-10-25 | GSE95412 | GEO