Mammalian Pumilio Proteins Control Cellular Morphology, Migration, and Adhesion
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ABSTRACT:
PROVIDER: PRJNA857310 | ENA |
REPOSITORIES: ENA
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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:Chlamydomonas pumilio var pumilio Cytochromome oxydase 1 (COI)
| PRJEB43352 | ENA
Project description:The regulation of flocculation, surface adhesion and invasive growth in the fission yeast Schizosaccharomyces pombe has focused primarily at the transcriptional level, but little is known with regards to posttranscriptional control. Here, we identified the Pumilio protein Pfr1 as a novel posttranscriptional regulator of these processes. Deletion of pfr1+ prevented flocculation, surface adhesion and invasive growth under inducing conditions, while overexpression of pfr1+ was sufficient to trigger flocculation. The flocculent phenotype of pfr1+ overexpression was dependent on the presence of the Gsf2 flocculin, but not on the Mbx2, Cbf12 and Adn3 transcription factors. In addition, we used RNA immunoprecipitation and expression microarrays to identify pvg1+ and SPBPB7E8.01, which encode a galactose pyruvyltransferase and glycophosphatidylinositol membrane protein, respectively, as putative mRNA targets potentially degraded by Pfr1. The mRNAs of these genes were upregulated and downregulated in the pfr1 deletion and overexpression strains, respectively, and contained putative binding sites in the 3â-untranslated region. We also discovered that ccr4+ and ste13+, which encode components of the mRNA decay machinery, were required for these processes, but did not suppress the pfr1+ overexpression flocculent phenotype when deleted. This data suggest that these processes in S. pombe involve multiple posttranscriptional-regulatory pathways of which one requires Pfr1. We generated 2 overexpression microarrays with dye swap that were biological replicates, 2 deletion microarrys with dye swap. Mutants samples were compared to empty vector control or wild type. 1 RIP-chip array was generated with IP rna compared to total RNA from the sample.
2016-03-24 | E-GEOD-79537 | biostudies-arrayexpress
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 regulation of flocculation, surface adhesion and invasive growth in the fission yeast Schizosaccharomyces pombe has focused primarily at the transcriptional level, but little is known with regards to posttranscriptional control. Here, we identified the Pumilio protein Pfr1 as a novel posttranscriptional regulator of these processes. Deletion of pfr1+ prevented flocculation, surface adhesion and invasive growth under inducing conditions, while overexpression of pfr1+ was sufficient to trigger flocculation. The flocculent phenotype of pfr1+ overexpression was dependent on the presence of the Gsf2 flocculin, but not on the Mbx2, Cbf12 and Adn3 transcription factors. In addition, we used RNA immunoprecipitation and expression microarrays to identify pvg1+ and SPBPB7E8.01, which encode a galactose pyruvyltransferase and glycophosphatidylinositol membrane protein, respectively, as putative mRNA targets potentially degraded by Pfr1. The mRNAs of these genes were upregulated and downregulated in the pfr1 deletion and overexpression strains, respectively, and contained putative binding sites in the 3’-untranslated region. We also discovered that ccr4+ and ste13+, which encode components of the mRNA decay machinery, were required for these processes, but did not suppress the pfr1+ overexpression flocculent phenotype when deleted. This data suggest that these processes in S. pombe involve multiple posttranscriptional-regulatory pathways of which one requires Pfr1.
2016-03-24 | GSE79537 | GEO
Project description:Previously, we suggested a cytosolic role for the histone-methyltransferase Ezh2 in regulating lymphocyte activation, but molecular mechanisms underpinning this extra-nuclear function remained unclear. Here we show that Ezh2 regulates integrin-signaling and adhesion dynamics of neutrophils and dendritic cells. Ezh2 deficiency impaired integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted talin binding to F-actin and thereby promoted adhesion structure turnover. This regulatory effect was abolished by targeted disruption of Ezh2 interactions with Vav1. Our studies reveal a novel extra-nuclear function for Ezh2 in regulating adhesion dynamics with implications for leukocyte migration, immune responses and potentially pathogenic processes. Control and Ezh2-deficient bone marrow derived immature and mature dendritic cells were analyzed in triplicates
2015-03-09 | E-GEOD-60146 | biostudies-arrayexpress