Functional analysis of the microtubule-interacting transcriptome

Sharp, Judith A.; Plant, Joshua J.; Ohsumi, Toshiro K.; Borowsky, Mark; Blower, Michael D.

doi:10.1091/mbc.e11-07-0629

Cited by 70 publications

(67 citation statements)

References 68 publications

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“…In this report we have described a simple method to produce CSF-arrested egg extracts from X. tropicalis 19 and use this extract to study microtubule-associated RNAs 7 . The basic procedure for producing CSF-arrested egg extracts from X. tropicalis is the same as used for X. laevis with a few key differences.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to the many well-documented cases of RNA localization to control embryonic patterning, several studies have documented mRNAs that are localized to the site of action of the encoded protein. Prominent examples include localization of the β-actin 3 and Arp2/3 4 mRNAs to the leading edge of motile fibroblasts and localization of the mRNAs for many mitotic regulators to meiotic and mitotic spindles [5][6][7] .…”

Section: Introductionmentioning

confidence: 99%

“…Purification of pseudopodia from mouse fibroblasts identified a diverse group of localized mRNAs 9 . Work from our group using biochemical purification of microtubules from meiotic Xenopus egg extracts identified hundreds of mRNAs that copurify with the spindle 5,7 . Our work showed that the majority of microtubule-localized mRNAs encode proteins that function in control of mitosis, supporting the idea that mRNAs are localized to the site of action of the coded protein.…”

Section: Introductionmentioning

confidence: 99%

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

Sharp

Blower

2013

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Sharp

Blower

2013

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“…Microtubule-associated RNAs were shown to be enriched in transcripts involved in cell division and spindle formation. 44 Fractionation of mouse C2C12 myoblast cells and Drosophila S2 cells into cytosolic, membrane-associated, and insoluble fractions revealed transcripts enriched in each compartment (Fig. 2B).…”

Section: Rna-seq-based Approachesmentioning

confidence: 99%

Genomic analysis of RNA localization

2014

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The localization of mRNAs to specific subcellular sites is widespread, allowing cells to spatially restrict and regulate protein production, and playing important roles in development and cellular physiology. This process has been studied in mechanistic detail for several RNAs. However, the generality or specificity of RNA localization systems and mechanisms that impact the many thousands of localized mRNAs has been difficult to assess. In this review, we discuss the current state of the field in determining which RNAs localize, which RNA sequences mediate localization, the protein factors involved, and the biological implications of localization. For each question, we examine prominent systems and techniques that are used to study individual messages, highlight recent genome-wide studies of RNA localization, and discuss the potential for adapting other high-throughput approaches to the study of localization.

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“…Specifically, we sought to uncover the sequence and structural motifs responsible for mRNA localization to spindle microtubules, a phenomenon that has been observed in a wide variety of organisms (Raff et al 1990;Groisman et al 2000;Lambert and Nagy 2002;Blower et al 2007;Kingsley et al 2007;Lécuyer et al 2007;Eliscovich et al 2008;Rabinowitz and Lambert 2010;Sharp et al 2011). In the course of our investigations, we made the interesting observation that long, dsRNA added exogenously to X. laevis egg extract localizes to spindle microtubules.…”

Section: Introductionmentioning

confidence: 95%

Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis

Schweidenback

Emerman

Jambhekar

et al. 2014

RNA

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ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, doublestranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR.

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Functional analysis of the microtubule-interacting transcriptome

Cited by 70 publications

References 68 publications

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

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

Genomic analysis of RNA localization

Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis

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