15 research outputs found
Label-Free Quantitative Phosphoproteomics of the Fission Yeast <i>Schizosaccharomyces pombe</i> Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies
The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe
The Interplay of Cohesin and RNA Processing Factors: The Impact of Their Alterations on Genome Stability
Cohesin, a multi-subunit protein complex, plays important roles in sister chromatid cohesion, DNA replication, chromatin organization, gene expression, transcription regulation, and the recombination or repair of DNA damage. Recently, several studies suggested that the functions of cohesin rely not only on cohesin-related protein–protein interactions, their post-translational modifications or specific DNA modifications, but that some RNA processing factors also play an important role in the regulation of cohesin functions. Therefore, the mutations and changes in the expression of cohesin subunits or alterations in the interactions between cohesin and RNA processing factors have been shown to have an impact on cohesion, the fidelity of chromosome segregation and, ultimately, on genome stability. In this review, we provide an overview of the cohesin complex and its role in chromosome segregation, highlight the causes and consequences of mutations and changes in the expression of cohesin subunits, and discuss the RNA processing factors that participate in the regulation of the processes involved in chromosome segregation. Overall, an understanding of the molecular determinants of the interplay between cohesin and RNA processing factors might help us to better understand the molecular mechanisms ensuring the integrity of the genome
The Interplay of Cohesin and RNA Processing Factors: The Impact of Their Alterations on Genome Stability
Cohesin, a multi-subunit protein complex, plays important roles in sister chromatid cohesion, DNA replication, chromatin organization, gene expression, transcription regulation, and the recombination or repair of DNA damage. Recently, several studies suggested that the functions of cohesin rely not only on cohesin-related protein–protein interactions, their post-translational modifications or specific DNA modifications, but that some RNA processing factors also play an important role in the regulation of cohesin functions. Therefore, the mutations and changes in the expression of cohesin subunits or alterations in the interactions between cohesin and RNA processing factors have been shown to have an impact on cohesion, the fidelity of chromosome segregation and, ultimately, on genome stability. In this review, we provide an overview of the cohesin complex and its role in chromosome segregation, highlight the causes and consequences of mutations and changes in the expression of cohesin subunits, and discuss the RNA processing factors that participate in the regulation of the processes involved in chromosome segregation. Overall, an understanding of the molecular determinants of the interplay between cohesin and RNA processing factors might help us to better understand the molecular mechanisms ensuring the integrity of the genome
Synthesis and Anticancer Activity of Novel 9-<i>O</i>-Substituted Berberine Derivatives
Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative
Identification of proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1 in the fission yeast Schizosaccharomyces pombe
Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation
Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation
Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation
<div><p>Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.</p></div
Scheme for dual action of casein kinase 1 (Hhp) on cohesin subunits to regulate meiotic chromosome segregation via Rec8 phosphorylation and to promote linear element formation, meiotic DSB formation, and recombination via Rec11 phosphorylation.
<p>Cohesin subunits Rec8 and Rec11 are loaded onto chromosomes prior to premeiotic replication, during which sister chromatid cohesion is established. Before, during, or after loading they are phosphorylated by casein kinase homologs Hhp1 and Hhp2. <i>Right</i>: Phosphorylation (P’n) of Rec11 leads to loading of the linear element (LinE) proteins Rec10, Rec25, Rec27, and Mug20, which in turn activate Rec12 (Spo11 homolog) and its partner proteins for DSB formation; DSBs lead to crossovers, which allow segregation of homologous centromeres, not sister centromeres, at the first meiotic division (MI). <i>Left</i>: Phosphorylation of Rec8 (generating P-Rec8) allows its cleavage, first in the arms, which allows segregation of homologous centromeres at MI; later, cleavage of Rec8 in the centromeres allows segregation of sister centromeres at MII. Rec8 is also needed for loading of Rec11 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.s011" target="_blank">S10 Fig</a>) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref042" target="_blank">42</a>] and is thus indirectly required for DSB formation and recombination [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref014" target="_blank">14</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref018" target="_blank">18</a>]. The precise timing of Rec8 and Rec11 phosphorylation is unknown. Rec8 and Rec11 (STAG3 functional homolog) and their phosphorylation may play similar roles in mammalian gametogenesis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#sec009" target="_blank">Discussion</a>).</p
Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe
Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe
Meiotic DNA breakage in <i>hhp</i>, <i>rec11</i>, and <i>rec8</i> mutants.
<p>Strains with the indicated mutations were induced for meiosis in the absence of an ATP analog. At the indicated times, DNA was extracted and analyzed by Southern blot hybridization. All time points for each mutant were run on the same gel, one gel for the two <i>Not</i>I fragments and another for the <i>ade6-3049</i> fragment (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.s002" target="_blank">S1 Fig</a>). Data below each lane with meiotically induced DNA are the percent of total DNA in the bands labeled a—f after subtraction of the intensity in the corresponding 0 hr lane. These data reflect DNA breakage at DSB hotspots. DSBs at the <i>ade6-3049</i> hotspot are spread over about 1 kb, indicated by the bar (f) on the right. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.s002" target="_blank">S1 Fig</a>. (A) The 501 kb <i>Not</i>I fragment J on chromosome 1 was analyzed with a probe at its left end [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref060" target="_blank">60</a>]. (B) The 1.5 Mb <i>Not</i>I fragment C on chromosome 2 was analyzed with a probe near its left end [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref018" target="_blank">18</a>]. (C) The 6.6 kb AflII fragment containing <i>ade6</i> on chromosome 3 was analyzed with a probe at its right end [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005225#pgen.1005225.ref061" target="_blank">61</a>].</p