scholarly journals An RNA-binding tropomyosin recruits kinesin-1 dynamically to oskar mRNPs

2016 ◽  
Author(s):  
Imre Gáspár ◽  
Vasily Sysoev ◽  
Artem Komissarov ◽  
Anne Ephrussi
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Localization ◽  
Posterior Pole ◽  
Transport Processes ◽  
Nurse Cells ◽  
Motor Activation ◽  
Cargo Transport ◽  
Dependent Transport ◽  
Localization Element

AbstractLocalization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. We show that the posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3’UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1 and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optmized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo associated dynein and kinesin-1.

scholarly journals Isolation of a Ribonucleoprotein Complex Involved in mRNA Localization in Drosophila Oocytes

2000 ◽  
Vol 148 (3) ◽  
pp. 427-440 ◽  
Author(s):  
James E. Wilhelm ◽  
Jennifer Mansfield ◽  
Nora Hom-Booher ◽  
Shengxian Wang ◽  
Christoph W. Turck ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mrna Localization ◽  
Posterior Pole ◽  
Nurse Cells ◽  
Embryonic Patterning ◽  
Large Protein ◽  
Genetic Studies ◽  
Large Protein Complex ◽  
Cold Shock Domain

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu–Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.

Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles of Drosophila: time-lapse analysis using video-enhanced contrast microscopy

1994 ◽  
Vol 107 (4) ◽  
pp. 849-858 ◽  
Author(s):  
J. Bohrmann ◽  
K. Biber
Keyword(s):  
Cytochalasin B ◽  
Time Lapse ◽  
Transport Processes ◽  
Nurse Cells ◽  
Previtellogenic Oocyte ◽  
Microtubule Inhibitors ◽  
Developmental Factors ◽  
Ring Canals ◽  
Dependent Transport

In Drosophila oogenesis, several morphogenetic determinants and other developmental factors synthesized in the nurse cells have been shown to accumulate in the oocyte during pre- to mid-vitellogenic stages. However, the mechanisms of the involved intercellular transport processes that seem to be rather selective have not been revealed so far. We have investigated in vitro, by means of video-enhanced contrast time-lapse microscopy, the transport of cytoplasmic particles from the nurse cells through ring canals into the oocyte during oogenesis stages 6–10A. At stage 7, we first observed single particles moving into the previtellogenic oocyte. The particle transfer was strictly unidirectional and seemed to be selective, since only some individual particles moved whereas other particles lying in the vicinity of the ring canals were not transported. The observed transport processes were inhibitable with 2,4-dinitrophenol, cytochalasin B or N-ethylmaleimide, but not with microtubule inhibitors. At the beginning of vitellogenesis (stage 8), the selective translocation of particles through the ring canals became faster (up to 130 nm/second) and more frequent (about 1 particle/minute), whereas during mid-vitellogenesis (stages 9–10A) the velocity and the frequency of particle transport decreased again. Following their more or less rectilinear passage through the ring canals, the particles joined a circular stream of cytoplasmic particles in the oocyte. This ooplasmic particle streaming started at stage 6/7 with velocities of about 80 nm/second and some reversals of direction at the beginning. The particle stream in the oocyte was sensitive to colchicine and vinblastine, but not to cytochalasin B, and we presume that it reflects the rearrangement of ooplasmic microtubules described recently by other authors. We propose that during stages 7–10A, a selective transport of particles into the oocyte occurs through the ring canal along a polarized scaffold of cytoskeletal elements in which microfilaments are involved. This transport might be driven by a myosin-like motor molecule. Either attached to, or organized into, such larger particles or organelles, specific mRNAs and proteins might become selectively transported into the oocyte.

Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1201-1211 ◽  
Author(s):  
L. Liang ◽  
W. Diehl-Jones ◽  
P. Lasko
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Cell Formation ◽  
Point Mutations ◽  
Posterior Pole ◽  
Nurse Cells ◽  
Protein Protein Interactions ◽  
Dead Box ◽  
Pole Plasm

The Drosophila gene vasa encodes a DEAD-box protein, which is localized during early oogenesis to the perinuclear region of the nurse cells and later to the pole plasm at the posterior end of the oocyte. Posterior localization of vasa protein depends upon the functions of four genes: capu, spir, osk and stau. We have found that localization of vasa to the perinuclear nuage is abolished in most vas alleles, but is unaffected by mutations in four genes required upstream for its pole plasm localization. Thus localization of vasa to the nuage particles is independent of the pole plasm assembly pathway. Furthermore, electron-dense nuage particles are less abundant in the cytoplasm of nurse cells from vas mutants that fail to exhibit perinuclear localization, suggesting that the formation of the nuage depends upon vas function. Eight of nine vas point mutations cause codon substitutions in a region conserved among DEAD-box genes. The proteins from two mutant alleles that retain the capacity to localize to the posterior pole of the oocyte, vasO14 and vasO11, are both severely reduced in RNA-binding and -unwinding activity as compared to the wild-type protein on a variety of RNA substrates including in vitro synthesized pole plasm RNAs. Initial recruitment of vasa to the pole plasm must consequently depend upon protein-protein interactions but, once localized, vasa must bind to RNA to mediate germ cell formation.

scholarly journals Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

2000 ◽  
Vol 20 (23) ◽  
pp. 8767-8782 ◽  
Author(s):  
Jin Ho Yoon ◽  
Dona C. Love ◽  
Anjan Guhathakurta ◽  
John A. Hanover ◽  
Ravi Dhar
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Synthetic Lethality ◽  
Sequence Similarity ◽  
Mrna Export ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Multicopy Suppressor ◽  
Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

scholarly journals Characterization of a beta-actin mRNA zipcode-binding protein.

1997 ◽  
Vol 17 (4) ◽  
pp. 2158-2165 ◽  
Author(s):  
A F Ross ◽  
Y Oleynikov ◽  
E H Kislauskis ◽  
K L Taneja ◽  
R H Singer
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Nuclear Export Signal ◽  
Degenerate Primers ◽  
Band Shift ◽  
Beta Actin ◽  
Actin Mrna

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

scholarly journals RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton

Cytoskeleton ◽  
2017 ◽  
Vol 74 (4) ◽  
pp. 161-169 ◽  
Author(s):  
L. A. Mamon ◽  
V. R. Ginanova ◽  
S. F. Kliver ◽  
A. O. Yakimova ◽  
A. A. Atsapkina ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins

scholarly journals Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

2005 ◽  
Vol 393 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Catherine Martel ◽  
Paolo Macchi ◽  
Luc Furic ◽  
Michael A. Kiebler ◽  
Luc Desgroseillers
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Nuclear Trafficking ◽  
Binding Domains ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

The extracellular electrical current pattern and its variability in vitellogenic Drosophila follicles

1986 ◽  
Vol 81 (1) ◽  
pp. 189-206 ◽  
Author(s):  
J. Bohrmann ◽  
A. Dorn ◽  
K. Sander ◽  
H. Gutzeit
Keyword(s):  
Juvenile Hormone ◽  
Current Density ◽  
Developmental Stages ◽  
Electrical Current ◽  
Posterior Pole ◽  
The Other ◽  
Follicular Epithelium ◽  
Nurse Cells ◽  
Vibrating Probe ◽  
Current Pattern

We determined the extracellular electrical current pattern around Drosophila follicles at different developmental stages (7–14) with a vibrating probe. At most stages a characteristic pattern can be recognized: current leaves near the oocyte end of the follicle and enters at the nurse cells. Only at late vitellogenic stages was an inward-directed current located at the posterior pole of many follicles. Most striking was the observed heterogeneity both in current pattern and in current density between follicles of the same stage. Different media (changed osmolarity or pH, addition of cytoskeletal inhibitors or juvenile hormone) were tested for their effects on extrafollicular currents. The current density was consistently influenced by the osmolarity of the medium but not by the other parameters tested. Denuded nurse cells (follicular epithelium locally stripped off) show current influx, while an accidentally denuded oocyte produced no current. Our results show that individual follicles may be electrophysiologically different, though their uniform differentiation during vitellogenesis does not reflect such heterogeneity.

Role for mRNA localization in translational activation but not spatial restriction of nanos RNA

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 659-669 ◽  
Author(s):  
S.E. Bergsten ◽  
E.R. Gavis
Keyword(s):  
Translational Control ◽  
Rna Localization ◽  
Early Embryo ◽  
Posterior Pole ◽  
Translational Repression ◽  
Control Element ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Body Patterning ◽  
Localization Signal

Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3′ untranslated region (3′UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3′UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3′UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.

scholarly journals Matrin3: Disorder and ALS Pathogenesis

2022 ◽  
Vol 8 ◽  
Author(s):  
Ahmed Salem ◽  
Carter J. Wilson ◽  
Benjamin S. Rutledge ◽  
Allison Dilliott ◽  
Sali Farhan ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Nuclear Dna ◽  
Neurodegenerative Disorder ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disordered Regions

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.

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