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.

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.

Gratuitous mRNA localization in the Drosophila oocyte

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 3013-3021 ◽  
Author(s):  
T.L. Serano ◽  
R.S. Cohen
Keyword(s):  
Protein Targeting ◽  
Mrna Localization ◽  
Posterior Pole ◽  
Dorsoventral Patterning ◽  
Mrna Transport ◽  
Anterior Cortex ◽  
Control Pattern ◽  
Or Gene ◽  
Pattern Forming

Many of the genes that control pattern formation in Drosophila encode mRNAs that are localized to discrete regions of the oocyte during oogenesis. While such localization is generally assumed to be important for the pattern-forming activities of these genes, this has been rigorously demonstrated in only a few cases. Here we address the role of mRNA localization for the dorsoventral patterning gene K10. K10 mRNA is localized to the oocyte's anterior cortex following its transport into the cell during early stages of oogenesis. We show that mutations in cappuccino and spire, which permit K10 mRNA transport, but prevent subsequent anterior localization, do not disrupt the synthesis or localization of K10 protein. We also show that modified K10 transgenes that produce transcripts which are uniformly distributed throughout the oocyte, or which are mislocalized to the oocyte's posterior pole, produce localized and functional K10 protein. We conclude that the anterior localization of K10 mRNA is not important for K10 protein targeting or gene function. We propose that the anterior localization of K10, and probably other mRNAs, is a by-product of mRNA transport and does not necessarily reflect a requirement for localization per se.

A staufen-like RNA-binding protein in translocation channels linking nurse cells to oocytes in Notonecta shows nucleotide-dependent attachment to microtubules

1999 ◽  
Vol 112 (17) ◽  
pp. 2947-2955
Author(s):  
S. Hurst ◽  
N.J. Talbot ◽  
H. Stebbings
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Nurse Cells ◽  
Double Stranded Rna ◽  
Standard Procedures ◽  
Rna Probes ◽  
Peptide Antibody

In Drosophila melanogaster the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that staufen associates with a motor protein which generates the movement. We have investigated this possibility using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a staufen peptide antibody and RNA probes we have identified a staufen-like protein, which specifically binds double-stranded RNA, in the nutritive tubes of Notonecta. We show that while the staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. We also show that the staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the staufen-like protein correlates with that of a kinesin protein within the ovaries. Our findings indicate that the staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor.

scholarly journals A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.

1997 ◽  
Vol 17 (8) ◽  
pp. 4852-4858 ◽  
Author(s):  
M M Kasten ◽  
S Dorland ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Gel Filtration ◽  
Transcriptional Regulators ◽  
Dna Binding Proteins ◽  
Multiprotein Complex ◽  
Large Protein ◽  
Genetic Studies ◽  
Large Protein Complex

The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae. Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins. We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da. Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes. The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase. We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression. Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

scholarly journals Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

2013 ◽  
Vol 24 (5) ◽  
pp. 566-577 ◽  
Author(s):  
Daniel Richmond ◽  
Raed Rizkallah ◽  
Fengshan Liang ◽  
Myra M. Hurt ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Biological Significance ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Large Protein ◽  
Large Protein Complex ◽  
The Stability ◽  
Mutant Cells

In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore–microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.

Multiple RNA regulatory elements mediate distinct steps in localization of oskar mRNA

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 169-178 ◽  
Author(s):  
J. Kim-Ha ◽  
P.J. Webster ◽  
J.L. Smith ◽  
P.M. Macdonald
Keyword(s):  
Pattern Formation ◽  
Untranslated Region ◽  
Anterior Margin ◽  
Regulatory Elements ◽  
Mrna Localization ◽  
Posterior Pole ◽  
Nurse Cells ◽  
Cis Acting ◽  
Cytoplasmic Proteins ◽  
Body Patterning

Pattern formation in the early development of many organisms relies on localized cytoplasmic proteins, which can be prelocalized as mRNAs. The Drosophila oskar gene, required both for posterior body patterning and germ cell determination, encodes one such mRNA. Localization of oskar mRNA is an elaborate process involving movement of the transcript first into the oocyte from adjacent interconnected nurse cells and then across the length of the oocyte to its posterior pole. We have mapped RNA regulatory elements that direct this localization. Using a hybrid lacZ/oskar mRNA, we identify several elements within the oskar 3′ untranslated region that affect different steps in the process: the early movement into the oocyte, accumulation at the anterior margin of the oocyte and finally localization to the posterior pole. This use of multiple cis-acting elements suggests that localization may be orchestrated in a combinatorial fashion, thereby allowing localized mRNAs with ultimately different destinations to employ common mechanisms for shared intermediate steps.

scholarly journals Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression ofoskartranslation

2018 ◽  
Author(s):  
Annabelle Dold ◽  
Hong Han ◽  
Niankun Liu ◽  
Andrea Hildebrandt ◽  
Mirko Brüggemann ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Binding Protein ◽  
Protein A ◽  
Cell Formation ◽  
Posterior Pole ◽  
Embryonic Patterning ◽  
Translational Repressor ◽  
Translational Activation ◽  
Translational Activator

AbstractMakorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. InDrosophilamaternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation ofoskar. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and bindsosk3’ UTR in a region adjacent to A-rich sequences. This binding site overlaps with Bruno1 (Bru1) responsive elements (BREs), which regulateosktranslation. We observe increased association of the translational repressor Bru1 withoskmRNA upon depletion of Mkrn1, indicating that both proteins compete foroskbinding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries fromMkrn1females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activatingosktranslation by displacing Bru1 from its 3’ UTR.Author SummaryTo ensure accurate development of theDrosophilaembryo, proteins and mRNAs are positioned at specific sites within the embryo. Many of these proteins and mRNAs are produced and localized during the development of the egg in the mother. One protein essential for this process that has been heavily studied is Oskar (Osk), which is positioned at the posterior pole. During the localization ofoskmRNA, its translation is repressed by the RNA-binding protein Bruno1 (Bru1), ensuring that Osk protein is not present outside of the posterior where it is harmful. At the posterior pole,oskmRNA is activated through mechanisms that are not yet understood. In this work, we show that the conserved protein Makorin 1 (Mkrn1) is a novel factor involved in the translational activation ofosk. Mkrn1 binds specifically tooskmRNA in a region that overlaps with the binding site of Bru1, thus alleviating the association of Bru1 withosk. Moreover, Mkrn1 is stabilized by poly(A) binding protein, a translational activator that bindsoskmRNA in close proximity to Mkrn1. Our work thus helps to answer a long-standing question in the field, providing insight about the function of Mkrn1 and more generally into embryonic patterning in animals.

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.

Profilin is required for posterior patterning of the Drosophila oocyte

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 2109-2116 ◽  
Author(s):  
L. Manseau ◽  
J. Calley ◽  
H. Phan
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoplasmic Streaming ◽  
Posterior Pole ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Nurse Cells ◽  
Actin Binding Protein ◽  
Mutant Phenotypes ◽  
Two Hybrid

We have investigated the role of the actin cytoskeleton during mid-oogenesis and have found that disrupting the actin cytoskeleton with cytochalasin D induces microtubule bundling and microtubule-based cytoplasmic streaming within the oocyte, similar to that which occurs prematurely in cappuccino and spire mutant oocytes. After examining a number of mutants that affect the actin cytoskeleton, we have found that chickadee, which encodes the actin-binding protein, profilin, shares this phenotype. In addition to the microtubule misregulation, mutants in chickadee resemble cappuccino in that they fail to localize STAUFEN and oskar mRNA to the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee. In an independent line of experiments, we have identified profilin as a CAPPUCCINO interactor in a two-hybrid screen for proteins that bind to CAPPUCCINO. This, together with the similarity of mutant phenotypes, suggests that profilin and CAPPUCCINO may interact during development.

Sign in / Sign up

Export Citation Format

Share Document