The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression

Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11, PHO12, and PHO84 is up-regulated in src1Δ cells. Notably, these PHO genes are located in subtelomeric regions of chromatin and exhibit a perinuclear location in vivo. Src1 spans the nuclear membrane twice and exposes its N and C domains with putative DNA-binding motifs to the nucleoplasm. Genome-wide chromatin immunoprecipitation–on-chip analyses indicated that Src1 is highly enriched at telomeres and subtelomeric regions of the yeast chromosomes. Our data show that the inner nuclear membrane protein Src1 functions at the interface between subtelomeric gene expression and TREX-dependent messenger RNA export through the nuclear pore complexes

Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity

Host Cell Phosphatidylcholine Is a Key Mediator of Malaria Parasite Survival during Liver Stage Infection

During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.Seventh Framework Programme (European Commission) (Grant Agreement 311502)Fundacao para a Ciencia e a Tecnologia (Grant EXCL/IMI-MIC/0056/2012)Fundacao para a Ciencia e a Tecnologia (Grant PTDC/IMI-MIC/1568/2012

(A and B) Relative increases (/wt, when wt) or decreases (wt/, when wt) of gene expression were plotted versus their distance to the closest telomere (A) or centromere (B)

A sliding window of 100 genes was used. The average of the 100 genes was used for the y axis, and the distance of the central gene in the window was used for the x axis. The bottom panel of A is an expanded view of the top graph showing the ∼27-kb region close to the telomeres, which exhibits a misregulation in the deletion strain. (C) Expression levels of mRNAs in wt and Δ cells. Total RNA of wt and Δ cells grown in HP and LP was prepared, and cDNA was analyzed by quantitative RT-PCR using specific primers for , , and . Each gene was assayed in triplicates. The mRNA levels of wt HP expression are set as one. One representative dataset of five times independently isolated RNA is shown. Error bars represent SD.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A) Schematic overview of pre-mRNA, mRNA, and protein products upon alternative splicing

Either a 126- or a 130-nt intron can be excised by using two alternative 5′ splice sites. In the latter case, a frame shift results in an earlier stop codon and, therefore, in a shorter protein with a different amino acid sequence at the C terminus compared with Src1-L. Conserved domains (HEH/LEM and MSC) and transmembrane domains (M) are indicated. Numbers represent amino acid residues. (B) Whole cell lysates of N- (TAP-Src1) and C-terminal TAP-tagged Src1-L or Src1-S were analyzed by SDS-PAGE followed by Western blotting using anti-ProtA antibodies. (C) Genetic relationship of splice variants with TREX–THO and TREX-2 components. The double-disruption strains were transformed with empty vector, GFP-Src1 splice variants, and the respective TREX component. Transformants were spotted in 10-fold serial dilutions on 5-FOA–containing plates for 5 d at the indicated temperatures.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A) The double-disrupted strains were transformed with the respective plasmid-borne wt or mutant genes

Growth was analyzed by spotting transformants in 10-fold serial dilutions on 5-FOA–containing plates at the indicated temperature for 5 d or on synthetic dextrose complete–Leu-Trp for 3 d (Δ, , and ). No growth indicates synthetic lethality. (B) Schematic representation of the genetic network between and factors involved in transcription-coupled mRNA export. Arrows to gray components indicate synthetic lethality/enhancement, and proteins depicted in white are genetically not linked to .<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

Design and Evaluation of Primaquine-Artemisinin Hybrids as a Multistage Antimalarial Strategy▿†

It is widely accepted that the struggle against malaria depends on the development of new strategies to fight infection. The “magic bullet” thought to be necessary to reach eradication should not only provide treatment for all Plasmodium spp. that infect human red blood cells but should also eliminate the replicative and dormant liver forms of the parasite. Moreover, these goals should ideally be achieved by using different mechanisms of action so as to avoid the development of resistance. To that end, two hybrid molecules with covalently linked primaquine and artemisinin moieties were synthesized, and their effectiveness against the liver and blood stages of infection was compared in vitro and in vivo with those of the parent compounds. Both hybrids displayed enhanced in vitro activities, relative to those of the parent compounds, against Plasmodium berghei liver stages. Both compounds were about as potent as artemisinin against cultured Plasmodium falciparum (50% inhibitory concentration [IC50], ∼10 nM). When used to treat a murine P. berghei infection, one of the molecules displayed better efficacy than an equimolar mixture of the parent pharmacophores, leading to improved cure and survival rates. These results reveal a novel approach to the design and evaluation of antimalarials based on the covalent combination of molecules acting on different stages of the parasite life cycle

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis