Alexandra Segref - Academia.edu (original) (raw)

Papers by Alexandra Segref

Molecular and cellular biology, 1998

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This co... more We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.

Cell Reports, 2014

In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configura... more In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging.

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindl... more Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells) 1,2 . The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown 3±5 , but it can be studied by adding chromatin beads to Xenopus egg extracts 6 . The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guaninenucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.

RNA, 2001

A Segref, I W Mattaj and M Ohno U snRNA export.

Nature Cell Biology, 2011

Protein ubiquitylation is a key post-translational control mechanism contributing to different ph... more Protein ubiquitylation is a key post-translational control mechanism contributing to different physiological processes, such as signal transduction and ageing. The size and linkage of a ubiquitin chain, which determines whether a substrate is efficiently targeted for proteasomal degradation, is determined by the interplay between ubiquitylation and deubiquitylation. A conserved factor that orchestrates distinct substrate-processing co-regulators in diverse species is the ubiquitin-selective chaperone CDC-48 (also known as p97). Several deubiquitylation enzymes (DUBs) have been shown to interact with CDC-48/p97, but the mechanistic and physiological relevance of these interactions remained elusive. Here we report a synergistic cooperation between CDC-48 and ATX-3 (the Caenorhabditis elegans orthologue of ataxin-3) in ubiquitin-mediated proteolysis and ageing regulation. Surprisingly, worms deficient for both cdc-48.1 and atx-3 demonstrated extended lifespan by up to 50%, mediated through the insulin-insulin-like growth factor 1 (IGF-1) signalling pathway. As lifespan extension specifically depends on the deubiquitylation activity of ATX-3, our findings identify a mechanistic link between protein degradation and longevity through editing of the ubiquitylation status of substrates involved in insulin-IGF-1 signalling.

Molecular Cell, 2002

Different classes of RNA are exported from the nucleus by distinct factors. We demonstrate that U... more Different classes of RNA are exported from the nucleus by distinct factors. We demonstrate that U1 snRNA is exported like an mRNA on insertion of a pre-mRNA intron or either sense or antisense mRNA exon sequences. mRNA-specific factors are recruited onto the spliced or elongated U1 RNA whereas U snRNA-specific factors are not, suggesting that an unstructured region of sufficient length in an RNA acts as a dominant determinant of mRNA identity. After export, spliced U1 RNA undergoes cytoplasmic maturation but is not reimported into the nucleus. These data provide insight into mechanisms for discrimination of different classes of nuclear RNA and demonstrate that two RNAs of identical sequence can have distinct cytoplasmic fates depending on their mode of export.

Molecular Cell, 2001

The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II tran... more The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II transcripts. It enhances the efficiency of several mRNA maturation steps and is essential for U snRNA nuclear export in multicellular eukaryotes. The 2A crystal structure of human CBC shows that the large subunit, CBP80, comprises three domains, each containing consecutive helical hairpins and resembling the so-called MIF4G domain found in several other proteins involved in RNA metabolism. The small subunit, CPB20, has an RNP fold and associates with the second and third domains of CBP80. Site-directed mutagenesis revealed 4 residues of CBP20 which are critical for cap binding. A model for cap binding is proposed based on these results and the known mode of binding of RNA to RNP domains.

Molecular Biology of the Cell, 2010

Coordination between cell fate specification and cell cycle control in multicellular organisms is... more Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein ␤-transducin repeat-containing protein (␤-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans ␤-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant. This article was published online ahead of print in MBC in Press

Genetics, 2011

In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis a... more In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis as it regulates the turnover of damaged proteins. However, it is still unclear how the UPS integrates intrinsic and environmental challenges to promote organismal development and survival. Here, we set up an in vivo degradation assay to facilitate the genetic identification of ubiquitin-dependent proteolysis pathways in the multicellular organism Caenorhabditis elegans. Using this assay, we found that mild induction of protein-folding stress, which is nontoxic for wild-type worms, strongly reduces ubiquitin-dependent protein turnover. Ubiquitin-mediated degradation is also reduced by metabolic stress, which correlates with life-span extension. Unlike other stress conditions, however, acute heat stress results in enhanced rather than reduced proteolysis. Intriguingly, our study provides the first evidence for the existence of tissue-specific degradation requirements because loss of key regulators of the UPS, such as proteasomal subunits, causes accumulation of the model substrate, depending on the tissue type. Thus, here we establish a screenable degradation assay that allows diverse genetic screening approaches for the identification of novel cell-type-specific proteostasis networks important for developmental processes, stress response, and aging, thereby substantially extending the work on recently described mechanistic UPS reporter studies.

EMBO reports, 2009

The nervous system coordinates many aspects of body function such as learning, memory, behaviour ... more The nervous system coordinates many aspects of body function such as learning, memory, behaviour and locomotion. Therefore, it must develop and maintain an intricate network of differentiated neuronal cells, which communicate efficiently with each other and with non-neuronal target cells. Unlike most somatic cells, differentiated neurons are post-mitotic and characterized by a highly polarized morphology that determines the flow of information. Among other post-translational modifications, the ubiquitination of specific protein substrates was recently shown to have a crucial role in the regulation of neuronal development and differentiation. Here, we review recent findings that illustrate the mechanisms that mediate the temporal and spatial control of neuronal protein turnover by the ubiquitin-proteasome system (UPS), which is crucial for the development and function of the nervous system.

Developmental Cell, 2005

Asymmetric distribution of cellular components underlies many biological processes, and the local... more Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.

Cell Metabolism, 2014

Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity,... more Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity, redox signaling, and apoptosis. Excessive levels of mitochondria-derived reactive oxygen species (ROS) promote mitochondrial dysfunction, triggering numerous metabolic disorders. However, the molecular basis for the harmful effects of excessive ROS formation is largely unknown. Here, we identify a link between mitochondrial stress and ubiquitindependent proteolysis, which supports cellular surveillance both in Caenorhabditis elegans and humans. Worms defective in respiration with elevated ROS levels are limited in turnover of a GFP-based substrate protein, demonstrating that mitochondrial stress affects the ubiquitin/proteasome system (UPS). Intriguingly, we observed similar proteolytic defects for disease-causing IVD and COX1 mutations associated with mitochondrial failure in humans. Together, these results identify a conserved link between mitochondrial metabolism and ubiquitindependent proteostasis. Reduced UPS activity during pathological conditions might potentiate disease progression and thus provides a valuable target for therapeutic intervention.

Cell, 1996

In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, ... more In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p-ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis.

Cell, 2000

In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Expo... more In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.

The American Journal of Human Genetics, 2012

Molecular and Cellular Biology, 2008

PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in me... more PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101: [187][188][189][190][191][192][193][194][195][196][197][198] 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation.