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1.
Int J Mol Sci ; 22(14)2021 Jul 15.
Article in English | MEDLINE | ID: mdl-34299194

ABSTRACT

A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the RUNX1-JAK2 fusion into one endogenous RUNX1 allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets-confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Hematopoietic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Janus Kinase 2/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/metabolism , STAT Transcription Factors/metabolism , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/genetics , Gene Expression Profiling/methods , Hematopoietic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Janus Kinase 2/genetics , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Signal Transduction
3.
Stem Cells Dev ; 27(19): 1376-1384, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30009677

ABSTRACT

Hematopoietic stem and progenitor cells (HSPCs) derived from human induced pluripotent stem cells (hiPSCs) hold great promise for disease modeling, drug screens, and eventually cell therapy approaches. During in vitro differentiation of hiPSCs into hematoendothelial progenitors, the emergence of CD34-positive cells indicates a critical step of lineage specification. To facilitate the monitoring of hematopoietic differentiation of hiPSCs, we established fluorescent reporter cells for the stem and progenitor cell marker CD34. An IRES-GFP (internal ribosome entry site green fluorescent protein) construct was introduced by CRISPR/Cas9 into the 3' untranslated region of one endogenous CD34 allele. Single-cell clones were generated after excision of the floxed puromycin resistance cassette by Cre recombination and correct insertion was confirmed by genotyping polymerase chain reaction and Southern blot. To validate their functionality, the reporter hiPSCs were in vitro differentiated toward CD34+ cells using the STEMdiff Hematopoietic Kit combined with short-term inhibition of GSK3 (glycogen synthase kinase 3). All cells expressing nuclear GFP were positive for cell surface CD34, thus allowing the direct monitoring of the differentiation of hiPSCs into CD34+ cells either by flow cytometry or confocal microscopy. After fluorescence-activated cell sorting, cells displaying high GFP expression exhibited increased colony-forming potential in the MethoCult colony-forming unit assays as compared with CD34+ cells obtained by magnetic-activated cell sorting. In summary, we have generated functional CD34 GFP reporter hiPSCs, which not only permit label-free separation of HSPCs, but also tracing of the emergence and fate of CD34+ progenitors at the single-cell level.


Subject(s)
Antigens, CD34/genetics , Colony-Forming Units Assay/methods , Green Fluorescent Proteins/genetics , Hematopoiesis , Hematopoietic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Antigens, CD34/metabolism , CRISPR-Cas Systems , Cells, Cultured , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Aging Cell ; 15(4): 744-54, 2016 08.
Article in English | MEDLINE | ID: mdl-27146333

ABSTRACT

Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor.


Subject(s)
Cell Differentiation , Cell-Derived Microparticles/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , MicroRNAs/metabolism , Osteogenesis , Adipose Tissue/cytology , Aging/blood , Cell-Derived Microparticles/ultrastructure , Cellular Senescence , Endothelial Cells/metabolism , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells/metabolism , Humans , MicroRNAs/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tetraspanin 30/metabolism
5.
Blood ; 125(8): 1282-91, 2015 Feb 19.
Article in English | MEDLINE | ID: mdl-25515960

ABSTRACT

PAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric protein has not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis.


Subject(s)
Janus Kinase 2/genetics , Oncogene Proteins, Fusion/genetics , PAX5 Transcription Factor/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Cell Death/drug effects , Cell Death/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression Regulation, Leukemic , HEK293 Cells , HeLa Cells , Humans , Janus Kinase 2/antagonists & inhibitors , Oncogene Proteins, Fusion/metabolism , Phosphorylation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Kinase Inhibitors/pharmacology , STAT Transcription Factors/metabolism , Transcriptome , Tumor Cells, Cultured
6.
Mol Cancer Res ; 12(4): 595-606, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24435167

ABSTRACT

UNLABELLED: PAX5, a transcription factor pivotal for B-cell commitment and maintenance, is one of the most frequent targets of somatic mutations in B-cell precursor acute lymphoblastic leukemia. A number of PAX5 rearrangements result in the expression of in-frame fusion genes encoding chimeric proteins, which at the N-terminus consistently retain the PAX5 DNA-binding paired domain fused to the C-terminal domains of a markedly heterogeneous group of fusion partners. PAX5 fusion proteins are thought to function as aberrant transcription factors, which antagonize wild-type PAX5 activity. To gain mechanistic insight into the role of PAX5 fusion proteins in leukemogenesis, the biochemical and functional properties of uncharacterized fusions: PAX5-DACH1, PAX5-DACH2, PAX5-ETV6, PAX5-HIPK1, and PAX5-POM121 were ascertained. Independent of the subcellular distribution of the wild-type partner proteins, ectopic expression of all PAX5 fusion proteins showed a predominant nuclear localization, and by chromatin immunoprecipitation all of the chimeric proteins exhibited binding to endogenous PAX5 target sequences. Furthermore, consistent with the presence of potential oligomerization motifs provided by the partner proteins, the self-interaction capability of several fusion proteins was confirmed. Remarkably, a subset of the PAX5 fusion proteins conferred CD79A promoter activity; however, in contrast with wild-type PAX5, the fusion proteins were unable to induce Cd79a transcription in a murine plasmacytoma cell line. These data show that leukemia-associated PAX5 fusion proteins share some dominating characteristics such as nuclear localization and DNA binding but also show distinctive features. IMPLICATIONS: This comparative study of multiple PAX5 fusion proteins demonstrates both common and unique properties, which likely dictate their function and impact on leukemia development.


Subject(s)
Leukemia/genetics , Leukemia/pathology , Oncogene Proteins, Fusion/genetics , PAX5 Transcription Factor/genetics , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , HEK293 Cells , HeLa Cells , Humans , Leukemia/metabolism , Microscopy, Confocal , Oncogene Proteins, Fusion/metabolism , PAX5 Transcription Factor/metabolism , Transcription, Genetic , Transfection
7.
Aging Cell ; 12(3): 446-58, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23496142

ABSTRACT

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21(CIP1) and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21(CIP) (1) , and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.


Subject(s)
Cell Proliferation , Cellular Senescence/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Apoptosis , Cells, Cultured , Cyclin-Dependent Kinase 2/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , Down-Regulation , Human Umbilical Vein Endothelial Cells/metabolism , Humans , NFI Transcription Factors/genetics , NFI Transcription Factors/metabolism , Neovascularization, Physiologic/genetics , RNA Interference , RNA, Small Interfering , Transfection , Up-Regulation , cdc25 Phosphatases/genetics
8.
Biochem J ; 438(1): 81-91, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21639856

ABSTRACT

The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin-proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEV(hPrp19/hPso4) and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.


Subject(s)
Alternative Splicing , Cell Nucleus/genetics , DNA Repair Enzymes/metabolism , Nuclear Proteins/metabolism , RNA Precursors/genetics , Spliceosomes/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Blotting, Western , Cell Nucleus/metabolism , DNA Repair Enzymes/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Protein Binding , RNA Splicing Factors , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Two-Hybrid System Techniques
9.
Epigenetics ; 6(1): 4-8, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20818169

ABSTRACT

Several recent publications demonstrate a co-activator function for a subgroup of plant homeodomain fingers, which in humans comprises PHF2, PHF8 and KIAA1718. Besides an N-terminal plant homeodomain (PHD) these proteins also harbor an enzymatically active Jumonji-C domain (JmjC). While they have been shown to bind via their PHDs to H3K4me3-bearing nucleosomes at active gene promoters, their JmjC-domains are able to remove mono- and dimethyl-lysine 9 or 27 on histone H3, and monomethyl-lysine 20 on histone H4, chromatin modifications which correlate with transcriptional repression. Such dual histone crosstalk insures the proper removal of repressive histone marks following transcriptional activation by RNA polymerases I and II. Mutations in the PHF8 gene lead to X-linked mental retardation (XLMR) and knockdown of KIAA1718 and PHF8 homologs in zebrafish causes brain defects. Thus, the co-activator function of this new class of chromatin modifying enzymes has important functional roles in neuronal development. To continue with the nomenclature for histone demethylases, we propose the usage of KDM7A, -B and -C for KIAA1718, PHF8 and PHF2 proteins, respectively.


Subject(s)
Chromatin/metabolism , Homeodomain Proteins/metabolism , Plant Proteins/metabolism , Promoter Regions, Genetic/physiology , Transcription, Genetic/physiology , Animals , Chromatin/genetics , Histones/genetics , Histones/metabolism , Homeodomain Proteins/genetics , Humans , Mutation , Plant Proteins/genetics , Zebrafish/genetics , Zebrafish/metabolism
10.
Mol Cell Biol ; 30(13): 3286-98, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20421419

ABSTRACT

Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.


Subject(s)
Histone Demethylases/metabolism , Histones/metabolism , RNA Polymerase II/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Cell Line , Gene Expression Profiling , Gene Knockdown Techniques , Histone Demethylases/genetics , Humans , Lysine/metabolism , Methylation , Microarray Analysis , Molecular Sequence Data , RNA Polymerase II/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics
11.
Aging Cell ; 9(2): 291-6, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20089119

ABSTRACT

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8(+) T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8(+) T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.


Subject(s)
Aging , Down-Regulation , MicroRNAs/genetics , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Humans , Oligonucleotide Array Sequence Analysis
12.
J Biol Chem ; 284(42): 29193-204, 2009 Oct 16.
Article in English | MEDLINE | ID: mdl-19641227

ABSTRACT

The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEV(Prp19-Pso4) complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEV(Prp19-Pso4), is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7alpha. Blom7alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7alpha overexpression. Thus we suggest that Blom7alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEV(Prp19-Pso4) complex at the splice sites.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Alternative Splicing , Animals , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Dose-Response Relationship, Drug , Escherichia coli/genetics , HeLa Cells , Humans , Introns , Protein Binding , Protein Structure, Tertiary , RNA Precursors/metabolism , Saccharomyces cerevisiae/genetics , Two-Hybrid System Techniques
13.
Stem Cells Dev ; 17(2): 355-66, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18447650

ABSTRACT

The quality of hematopoietic stem cells (HSCs) is essentially defined by two characteristics, i.e., multilineage differentiation and self-renewal capacity. Thus, it is of high priority to clarify mechanisms that regulate these functions and to understand them at the molecular level. In the present study, we investigated the role of senescence evasion factor (synonymously hPrp19,hPSO4,hNMP200: SNEV), a multifunctional protein involved in pre-mRNA splicing, regulation of replicative life span, and DNA repair. Here we report that murine SNEV mRNA expression is high in lineage-depleted (Lin(-)) precursor cells of the bone marrow immediately after isolation as compared to fully differentiated peripheral blood lymphocytes (PBLs). Furthermore, the progenitor cell subset with highest colony-forming ability and self-renewal capacity (Lin(-), Sca-1(+)) showed also the highest SNEV expression. To test if the observed differences in SNEV mRNA levels cause stem cell defects, Lin(-) cells derived from heterozygous SNEV knockout mice were tested for primary as well as secondary colony-forming potential as a measure of self-renewal capacity. Interestingly, both, primary and secondary colonies were significantly less formed from SNEV(+/-) cells, a defect that was rescued by ectopic SNEV expression. Similarly, bone marrow cells derived from the short-lived Senescence-Accelerated-Mouse-Prone (SAMP8) model showed similar differences in comparison to the aging-resistant (SAMR1) control strain. These data suggest that the expression of SNEV is closely associated with the growth of murine HSCs and determines the proliferative and repopulating capacity of phenotypically defined HSC subsets.


Subject(s)
Allelic Imbalance/physiology , Hematopoietic Stem Cells/physiology , Nuclear Matrix-Associated Proteins/genetics , Animals , Blood Cells/metabolism , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Female , Hematopoietic Stem Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Matrix-Associated Proteins/metabolism , RNA Splicing Factors
14.
Mol Cell Biol ; 27(8): 3123-30, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17283042

ABSTRACT

SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.


Subject(s)
Embryo, Mammalian/pathology , Nuclear Matrix-Associated Proteins/deficiency , Nuclear Proteins/deficiency , Animals , Blastocyst/cytology , Cell Proliferation , Crosses, Genetic , Female , Fetal Death , Fibroblasts/cytology , Gene Expression Regulation , Gene Targeting , Heterozygote , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Splicing Factors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, Protein
15.
Nucleic Acids Res ; 33(21): 6868-83, 2005.
Article in English | MEDLINE | ID: mdl-16332694

ABSTRACT

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human-yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein-protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56-74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.


Subject(s)
RNA Splicing , Spliceosomes/metabolism , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Cell Nucleus/chemistry , Conserved Sequence , DNA Repair Enzymes , Evolution, Molecular , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Nuclear Proteins , Peptides/pharmacology , Phenotype , Protein Structure, Tertiary , RNA Precursors/metabolism , RNA Splicing/drug effects , RNA Splicing Factors , RNA, Messenger/metabolism , RNA-Binding Proteins/analysis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Spliceosomes/drug effects , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics
16.
Exp Cell Res ; 309(1): 121-36, 2005 Sep 10.
Article in English | MEDLINE | ID: mdl-15964568

ABSTRACT

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.


Subject(s)
Cell Line, Transformed/physiology , Cellular Senescence/physiology , DNA-Binding Proteins , Endothelial Cells/cytology , Endothelial Cells/physiology , Gene Expression Regulation , Telomerase , Cell Differentiation/physiology , Cell Division/physiology , Cell Line , DNA-Binding Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Endothelial Cells/pathology , Humans , Polymerase Chain Reaction , Proteomics , Telomerase/genetics , Transfection
17.
Biochem J ; 388(Pt 2): 593-603, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15660529

ABSTRACT

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.


Subject(s)
Cysteine Endopeptidases/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans , Carrier Proteins/chemistry , Cell Line , Conserved Sequence , Cysteine Endopeptidases/physiology , DNA Repair Enzymes , Evolution, Molecular , Humans , Molecular Sequence Data , Nuclear Proteins , Proteasome Endopeptidase Complex/chemistry , Protein Binding , Protein Conformation , RNA Splicing Factors , Sequence Homology, Amino Acid , Signal Transduction , Ubiquitin-Protein Ligases/physiology
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