69 research outputs found

    Aberrant mRNA transcripts and nonsense-mediated decay

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    Nobody's perfect, and even the cell turns out a certain fraction of erroneous mRNA transcripts. One of the key quality control mechanisms put in place to recognize and eliminate these transcripts before they can be translated into faulty proteins is nonsense-mediated decay. Proteins involved in nonsense-mediated decay are highly conserved across species from plants to humans, and recent studies in Arabidopsis thaliana reveal both intriguing similarities and differences in the mechanisms employed to carry it out

    Recent advances in proximity-based labeling methods for interactome mapping [version 1; referees: 2 approved]

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    Proximity-based labeling has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in the mapping of protein–protein interactions. Ongoing optimization of enzyme tags and delivery methods has improved both temporal and spatial resolution, and the technique has been successfully employed in numerous small-scale (single complex mapping) and large-scale (network mapping) initiatives. When paired with quantitative proteomic approaches, the ability of these assays to provide snapshots of stable and transient interactions over time greatly facilitates the mapping of dynamic interactomes. Furthermore, recent innovations have extended biotin-based proximity labeling techniques such as BioID and APEX beyond classic protein-centric assays (tag a protein to label neighboring proteins) to include RNA-centric (tag an RNA species to label RNA-binding proteins) and DNA-centric (tag a gene locus to label associated protein complexes) assays

    Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry

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    Label-based quantitative mass spectrometry analysis of affinity purified complexes, with its built-in negative controls and relative ease of use, is an increasingly popular choice for defining protein-protein interactions and multiprotein complexes. This approach, which differentially labels proteins/peptides from two or more populations and combines them prior to analysis, permits direct comparison of a protein pulldown (e.g. affinity purified tagged protein) to that of a control pulldown (e.g. affinity purified tag alone) in a single mass spectrometry (MS) run, thus avoiding the variability inherent in separate runs. The use of quantitative techniques has been driven in large part by significant improvements in the resolution and sensitivity of high-end mass spectrometers. Importantly, the availability of commercial reagents and open source identification/quantification software has made these powerful techniques accessible to nonspecialists. Benefits and drawbacks of the most popular labeling-based approaches are discussed here, and key steps/strategies for the use of labeling in quantitative immunoprecipitation experiments detailed

    The Cajal body and the nucleolus : “in a relationship” or “it's complicated”?

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    From their initial identification as 'nucleolar accessory bodies' more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the two structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.PostprintPeer reviewe

    Mitotic phosphatases: no longer silent partners

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    Recent work has highlighted the important role played by protein phosphatase complexes in the regulation of mitosis from yeast to mammals. There have been important advances in defining the roles of the protein serine/threonine phosphatases PP1 and PP2A and the dual specificity protein tyrosine phosphatases CDC25 and Cdc14. Three independent studies defined a regulatory role for PP2A in the control of sister chromatid cohesion, involving a direct interaction with shugoshin. A chromatin targeting subunit has been identified for PP1 and the complex shown to play an essential role in chromosome segregation. Key regulatory residues within CDC25 have been mapped and its activity tied both to the initial activation of cyclin-dependent kinases at the centrosome and to DNA damage checkpoints. Novel roles have been defined for Cdc14, including regulation of rDNA and telomere segregation and participation in spindle assembly. These exciting advances show that protein phosphatases are not merely silent partners to kinases in regulating the control of cell division. Introduction The process of cell division is complex and involves multiple independent regulatory steps, most of which are controlled by reversible protein phosphorylation. In higher eukaryotes, mitosis involves condensation of chromosomes, disassembly of the nuclear lamina, breakdown of the nuclear envelope and disassembly of many forms of nuclear bodies, including nucleoli. Completion of mitosis requires alignment and proper segregation of chromosomes into daughter cells followed by reassembly of nuclei and cytokinesis. These and many other events, such as centrosome separation and spindle assembly, are tightly regulated, and several critical checkpoints occur during mitosis to ensure fidelity. Failure to complete any of the key steps can trigger apoptosis and cell death. While the important role of protein phosphorylation in regulating mitotic events has long been recognized, much of the work in this area has focused on the kinases, primarily the Cdk/Cyclin, Aurora, Polo-like and NIMA families (see This review will focus on recent advances in understanding the contributions of four major classes of protein phosphatases to the regulation of processes involved in controlling cell division, specifically the protein serine/ threonine phosphatases PP1 and PP2A and the dualspecificity protein tyrosine phosphatases (DUSPs) CDC25 and Cdc14. We will draw on examples from species as diverse as yeast, insects and mammals, reflecting the high evolutionary conservation of these regulated events. Serine/threonine phosphatases Both PP1 (termed Glc7 in budding yeast and Dis2 in fission yeast) and PP2A are serine/threonine-specific protein phosphatase catalytic subunits that form holoenzyme complexes with one or more regulatory subunits. These regulatory subunits can affect cellular location and/or substrate specificity. In contrast with most kinases, the PP1 and PP2A catalytic subunits can potentially act on a wide range of substrates and thus substrate specificity is conferred by their interaction partners. Thus, a critical step in understanding the role of PP1 and PP2A holoenzymes is to define their regulatory subunits and the mechanism by which they are targeted to their physiological substrates. Much of the literature ascribing specific roles to PP1 or PP2A has relied on differential effects of inhibitors such as okadaic acid, which in vitro blocks PP2A activity at lower concentrations than are required to inhibit PP1 PP2A PP2A plays a prominent role in the regulation of mitosis and signalling pathways. In addition to its interaction with both scaffolding and variable subunits (termed 'A' and 'B' subunits, respectively) in a trimeric complex (see Using immunoprecipitation and yeast two-hybrid studies, several groups independently identified a specific PP2A trimeric complex that interacts with Sgo1 [7 ,8 ,9 ]. On the basis of RNAi studies and analysis of a non-PP2A-binding hSgo1 mutant, Tang and colleagues [7 ] proposed that interaction with PP2A is required for centromeric localization of hSgo1 and proper chromosome segregation. Independently, the same PP2A complex was immunopurified from HEK 293T cells using Flagtagged hSgo1 [8 ]. Immunofluorescence studies by Kitajima and colleagues showed colocalization of hSgo1 and the B56 PP2A regulatory subunit at mammalian centromeres. Using RNAi in mammals, they also reported that knockdown of hSgo2, but not of hSgo1, resulted in loss of centromeric PP2A. Conversely, knockdown of PP2A led to a loss of centromeric hSgo1 [8 ]. Studies on both budding and fission yeast undergoing meiosis also showed that Sgo1 interacts with PP2A at centromeres and serves to protect the cohesin Rec8 subunit from phosphorylation and cleavage [9 ]. Interestingly, tethering of yeast PP2A at specific sites on chromosome arms preserved cohesion at these sites even after meiosis I, when arm cohesin should dissociate, showing an intrinsic ability of PP2A to protect cohesin, independent of Sgo1 [8 ,9 ]. The PP2A complex may thus work both directly at centromeres to maintain cohesion and by facilitating accumulation of Sgo1, which also acts to prevent cleavage of cohesin. Taken together, these studies point to an important new role for PP2A in the control of chromosome cohesion, mediated, at least in part, through interactions with shugoshins ( PP2A has also been implicated in regulating mitotic exit. Wang and Ng [10] provided evidence suggesting that a PP2A-Cdc55 complex dephosphorylates the mitotic exit network (MEN) activator Tem1 in budding yeast. This prevents mitotic exit by blocking release of Cdc14 from 624 Cell division, growth and death Figure 1 Role of PP2A in maintenance of chromosome cohesion. This diagram summarizes three recent studies that identified a specific PP2A trimeric complex acting with shugoshin to protect cohesin at centromeres from phosphorylation and cleavage until the metaphase-anaphase transition. In metazoan mitosis, cohesin is removed from chromosome arms at prometaphase but remains at the centromere regions, protected by shugoshin and PP2A. At the metaphase-anaphase transition, separase is activated and cleaves this residual cohesin, resulting in a loss of cohesion and separation of sister chromatids. PP1 PP1 has been shown to contribute to the regulation of multiple cellular processes including glycogen metabolism and muscle contraction, mediated by interaction of the PP1 catalytic domain with regulatory proteins termed 'targeting subunits'. Over 50 have been described to date, and they have the potential to regulate both the localization and the catalytic activity of PP1 (see [14] for review). Most targeting subunits share a common 'RVXF' motif that mediates direct binding to PP1 An elegant series of experiments has described a role for PP1 in controlling nuclear envelope assembly at the end of mitosis [24][25][26]. When cells enter mitosis, nuclear lamina disassembly is promoted by phosphorylation of B-type lamins. AKAP149, an ER and nuclear membrane protein, was shown to target PP1 (via an RVXF motif) to dephosphorylate B-type lamins at telophase, enabling their polymerization and thus lamina reassembly. A short peptide from AKAP149 containing the RVXF motif can displace PP1 and induce mislocalization of B-type lamins to the cytoplasm. Although the cells were able to complete mitosis, they died by apoptosis within six hours, suggesting that disruption of lamin assembly may directly trigger apoptosis. The association of PP1 isoforms with centrosomes, kinetochores and the cellular cortex and midbody region (see Pinsky et al. [29 ] took advantage of the regulation of Glc7 by targeting subunits to explore its interaction with Ipl1 (Aurora B) in budding yeast. Glc7 is known to antagonize Ipl1 activity, but it was unclear whether it dephosphorylates its substrates or regulates the kinase directly. Although the targeting subunit has not been identified, titratation of Glc7 away from Ipl1 by overexpression of Glc7 binding proteins that do not play roles in chromosome segregation led to increased phosphorylation of Ipl1 substrates, leading the authors to propose that Glc7 acts to ensure accurate chromosome segregation by dephosphorylating Ipl1 targets. CDC25 CDC25 was first identified in fission yeast 20 years ago as a factor required for entry into mitosis [30]. It is now known to activate cyclin-dependent kinases (Cdks) by removing inhibitory phosphates, which leads to Cdk phosphorylation of multiple substrates that drive the cell division process forward (see Three mammalian genes were identified that complement the yeast cdc25 knockout strain. The proteins encoded by these genes, termed CDC25A, CDC25B and CDC25C, are 60% identical in their C-terminal regions, which include the catalytic core containing the CX 5 R motif common to all protein tyrosine phosphatases. In contrast to the reasonably high homology of their catalytic domains, they are only 20-25% identical in their N-terminal regulatory domains, which contain sites for various post-translational modifications and sitespecific protein interactions, including phosphorylation of key serine and threonine residues, ubiquitination, phosphorylation-dependent binding of 14-3-3 proteins and Pin1-dependent prolyl isomerization (see There is a dramatic hyperphosphorylation of CDC25 during the transition from interphase to mitosis, and several mitotic phosphorylation sites have been mapped (see While all three mammalian CDC25 phosphatases activate their Cdk substrates in the same manner, they appear to have distinct roles in regulating cell cycle transitions (see Mitotic phosphatases: no longer silent partners Trinkle-Mulcahy and Lamond 627 have not yet been ruled out. (b) The G 2 /M transition is regulated in a similar way, with CDC25 activating Cdk1/Cyclin B by dephosphorylating critical residues. All three mammalian CDC25 isoforms have been implicated in regulation of this pathway. (c) The initial activation of Cdk1/Cyclin B has been shown to occur at centrosomes as they begin to separate during prophase, and involves the phosphorylation and activation of CDC25B by the Ajuba-Aurora A complex. The divergent N-terminal regulatory domains of the three mammalian CDC25 isoforms contain a variety of regulatory sites, including phosphorylation sites, 14-3-3 binding sites, domains that regulate degradation and nuclear import and export signals. Several of these known and recently described regulatory sites have been summarized here for (d) CDC25A, (e) CDC25B and (f) CDC25C. Cdc14 While Cdc25 is a key regulator of initiation of mitosis (and hence DNA damage checkpoint control), Cdc14 is a key regulator of late mitotic events, coordinating the temporal and spatial control of chromosome segregation with mitotic spindle disassembly and cytokinesis. In the budding yeast S. cerevisiae, Cdc14p plays a key role in exit from mitosis by dephosphorylating Cdk targets (reviewed in FEAR-controlled release of Cdc14p in budding yeast is also important for division of nucleoli and resolution of highly repetitive rDNA and telomere regions, as demonstrated in two recent studies. These regions separate at mid-anaphase, long after cohesin is cleaved. D'Amours and colleagues 628 Cell division, growth and death Figure 4 Cross-species comparison of Cdc14 localization and function. Cdc14 homologues from four different eukaryotes are listed, showing their localization during interphase and throughout mitosis. Nuclei are shown in green, spindle pole bodies (centrosomes) in red, microtubules in pink and chromosomes in blue. The localization of Cdc14 at these sites is shown in yellow. Known mitotic functions for these homologues are also listed

    Repo-Man recruits PP1γ to chromatin and is essential for cell viability

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    Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1α and -γ are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1γ is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1γ onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1α and -γ in vivo. When overexpressed, Repo-Man can also recruit PP1α to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference–induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1γ and is required for the recruitment of PP1 to chromatin

    NOPdb: Nucleolar Proteome Database

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    The Nucleolar Proteome Database (NOPdb) archives data on >700 proteins that were identified by multiple mass spectrometry (MS) analyses from highly purified preparations of human nucleoli, the most prominent nuclear organelle. Each protein entry is annotated with information about its corresponding gene, its domain structures and relevant protein homologues across species, as well as documenting its MS identification history including all the peptides sequenced by tandem MS/MS. Moreover, data showing the quantitative changes in the relative levels of ∼500 nucleolar proteins are compared at different timepoints upon transcriptional inhibition. Correlating changes in protein abundance at multiple timepoints, highlighted by visualization means in the NOPdb, provides clues regarding the potential interactions and relationships between nucleolar proteins and thereby suggests putative functions for factors within the 30% of the proteome which comprises novel/uncharacterized proteins. The NOPdb () is searchable by either gene names, nucleotide or protein sequences, Gene Ontology terms or motifs, or by limiting the range for isoelectric points and/or molecular weights and links to other databases (e.g. LocusLink, OMIM and PubMed)

    Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

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    © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.Norwegian Cancer Society [3367910]; South-Eastern Norway Health Authorities [2014035, 2013017]; Norwegian Research Council [275918]; EEA Czech-Norwegian Research Programme (Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports [Project Contract no. MSMT-22477/2014 (7F14061)]. Funding for open access charge: Norwegian Research Council.info:eu-repo/semantics/publishedVersio

    Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

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    The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments
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