Association of ribosomes with in vitro assembled microtubules

Suprenant, Kathy A.; Tempero, Libeth B.; Hammer, Lorraine E.

doi:10.1002/cm.970140310

Cited by 44 publications

(49 citation statements)

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“…By electron microscopy, the MT-RNP complexes consisted of microtubule arrays studded with ribosomes that appeared to be connected via a salt-extractable, protease-sensitive stalk (Hamill et al 1994). Similar studding of ribosomes on MTs has been reported to occur in vivo along the MT arrays of the mitotic spindle (Hirokawa et al 1985;Suprenant et al 1989) and ciliary platform rootlets of epithelial cells at the primary mesenchyme blastula stage of sea urchin embryogenesis (Gibbins et al 1969).…”

Section: Introductionmentioning

confidence: 54%

“…The observation that seawi and ribosomes (Suprenant et al 1989) are associated with the microtubules of the mitotic spindle provides an avenue for spatial segregation and anchorage of RNP complexes within the developing embryo. Once spatially segregated, translational activation of localized and arrested complexes may require subsequent assembly and activation of the translational machinery as elegantly shown in Drosophila (Carrera et al 2000;Lipshitz and Smibert 2000;Harris and Macdonald 2001).…”

Section: Seawi and Its Potential Role In Sea Urchin Embryogenesismentioning

confidence: 99%

“…In Strongylocentrotus purpuratus eggs and two-cell embryos, a reconstituted microtubule-ribonucleoprotein (MT-RNP) complex consisting of ribosomes, microtubules, mRNA, 107-kDa major vault protein, 77-kDa EMAP, 80-and 66-kDa poly-A binding proteins, and an uncharacterized 100-kDa protein was isolated by temperature-and pHdependent polymerization cycling of MTs in cell extracts (Suprenant et al 1989;Hamill et al 1994;Hamill and Suprenant 1997). By electron microscopy, the MT-RNP complexes consisted of microtubule arrays studded with ribosomes that appeared to be connected via a salt-extractable, protease-sensitive stalk (Hamill et al 1994).…”

Section: Introductionmentioning

confidence: 99%

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Seawi—a sea urchin piwi/argonaute family member is a component of MT-RNP complexes

Rodríguez¹,

Seipel²,

Hamill³

et al. 2005

RNA

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The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic spindle during cell division, and the small micromeres where its levels were enriched during the early cleavage stage. Lastly, we discuss how seawi, as a piwi/argonaute family member, may play a fundamentally important role in sea urchin animal-vegetal axis formation and stem cell maintenance.

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Section: Introductionmentioning

confidence: 54%

Section: Seawi and Its Potential Role In Sea Urchin Embryogenesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Seawi—a sea urchin piwi/argonaute family member is a component of MT-RNP complexes

Rodríguez¹,

Seipel²,

Hamill³

et al. 2005

RNA

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“…Inasmuch as 1-tubulin mRNA is cotranslationally degraded, and the process is coupled to translational elongation (12), a reasonable possibility is that the cofactor is the ribosome itself. In this regard, it is interesting to note that ribosomes can associate with sea urchin microtubules in vitro (29), although the significance of this finding to tubulin autoregulation is obscure.…”

Section: Resultsmentioning

confidence: 99%

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Theodorakis¹,

Cleveland²

1992

Mol. Cell. Biol.

114

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Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a "-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of 1-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal 1-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the 13-tubulin nascent peptide selectively disrupts the regulation of 13-tubulin, but not a-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of "-tubulin mRNA mediated through binding of one or more cellular factors to the 13-tubulin nascent peptide.Microtubules are dynamic filamentous structures that participate in a wide variety of cellular processes, including mitosis, vesicle transport, and cellular motility. The principle component of microtubules is tubulin, a heterodimer of a and i subunits that exist in a state of dynamic equilibrium with the microtubule polymer. The synthesis of tubulin is tightly coupled to the state of polymerization of the microtubule. Increasing the cytoplasmic concentration of tubulin subunits, for example by depolymerizing microtubules with drugs (1,8) or by microinjection of unassembled tubulin subunits (9), leads to a rapid and specific arrest of the synthesis of a-and ,B-tubulin, caused by a decrease in the level of tubulin mRNAs (8).A number of observations have led to the conclusion that the modulation of tubulin synthesis in response to changes in tubulin subunit concentration is a posttranscriptional cytoplasmic event. First, run-on transcription in isolated nuclei reveals no changes in the transcription rates of tubulin genes when microtubules are depolymerized by colchicine (7). Second, the colchicine-induced decrease in tubulin synthesis rates occurs even in enucleated cells (5, 26). Finally, hybrid genes composed of tubulin-coding sequences and heterologous promoters still respond to changes in tubulin subunit levels in transfected cells (13,32,33).Previous efforts have shown that the sequence necessary and sufficient for the selective degradation of a ,B-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of 3-tubulin, MREI (32). Several lines of evidence indicate t...

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“…We have successfully used these extracts to identify microtubule-associated RNAs . Similar microtubule purification schemes have been described for many different organisms 25,26 , although the microtubule associated RNAs have not been examined. The approach described here could be used with any organism that can produce a concentrated cytoplasmic extract capable of nucleating microtubules.…”

Section: Discussionmentioning

confidence: 99%

Production of <em>Xenopus tropicalis</em> Egg Extracts to Identify Microtubule-associated RNAs

Sharp

Blower

2013

JoVE

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Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed highthroughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

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Association of ribosomes with in vitro assembled microtubules

Cited by 44 publications

References 50 publications

Seawi—a sea urchin piwi/argonaute family member is a component of MT-RNP complexes

Seawi—a sea urchin piwi/argonaute family member is a component of MT-RNP complexes

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Production of <em>Xenopus tropicalis</em> Egg Extracts to Identify Microtubule-associated RNAs

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