Limits...
Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.

Blower MD, Feric E, Weis K, Heald R - J. Cell Biol. (2007)

Bottom Line: We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis.Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes.Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. blower@molbio.mgh.harvard.edu

ABSTRACT
RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.

Show MeSH
Specific mRNAs are localized to microtubules in mitotic human extracts. (A) Mitotic extracts were prepared from synchronized human cells, and microtubule asters induced by the addition of taxol were fixed and spun onto coverslips and stained for RNA (using SYTO RNASelect) and tubulin. RNA was localized to microtubule asters in a granular staining pattern. (B) Asters from this extract were pelleted through a glycerol cushion and isolated RNA was run on an agarose gel and stained with ethidium bromide. rRNA and mRNA pelleted more efficiently from extracts when microtubules were polymerized. Pelleted nucleic acid diminished upon treatment with RNaseA. (C) MT-mRNA and total mRNA were hybridized to Affymetrix microarrays as described in Fig. 1. As seen with X. laevis, specific mRNAs were enriched on mitotic human microtubules, whereas the vast majority of mRNAs were not. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2373496&req=5

fig2: Specific mRNAs are localized to microtubules in mitotic human extracts. (A) Mitotic extracts were prepared from synchronized human cells, and microtubule asters induced by the addition of taxol were fixed and spun onto coverslips and stained for RNA (using SYTO RNASelect) and tubulin. RNA was localized to microtubule asters in a granular staining pattern. (B) Asters from this extract were pelleted through a glycerol cushion and isolated RNA was run on an agarose gel and stained with ethidium bromide. rRNA and mRNA pelleted more efficiently from extracts when microtubules were polymerized. Pelleted nucleic acid diminished upon treatment with RNaseA. (C) MT-mRNA and total mRNA were hybridized to Affymetrix microarrays as described in Fig. 1. As seen with X. laevis, specific mRNAs were enriched on mitotic human microtubules, whereas the vast majority of mRNAs were not. Bars, 5 μm.

Mentions: We next asked whether localization of specific mRNAs to microtubules during mitosis is conserved among different cell types and species. Synchronized HeLa cells were used to prepare mitotic extracts that assemble radial microtubule asters when treated with taxol, reproducing many aspects of mitotic microtubule organization (Gaglio et al., 1995; Mack and Compton, 2001). RNA localized in a granular pattern on taxol microtubule asters in HeLa extracts, and both rRNA and mRNA specifically copurified with microtubules (Fig. 2, A and B). To identify MT-mRNAs in mitotic HeLa extracts, we hybridized MT-RNA– derived cRNAs to Affymetrix microarrays and compared the abundance of mRNAs in the microtubule sample to the total extract. Similar to our results with X. laevis, we observed that there was a continuum of mRNA binding to microtubules, with ∼10% of all mRNAs enriched 1.5-fold or more on mitotic microtubules (Fig. 2 C), and that mRNAs encoding proteins involved in various aspects of mitosis and DNA metabolism (replication, repair, and topology) were overrepresented in the MT-mRNA fraction (Table S5). The CPE was also overrepresented in the UTRs of MT-mRNAs with 5.7% of them possessing this element, which is a 4.1-fold enrichment compared with all mRNAs (Table S3, available at http://www.jcb.org/cgi/content/full/jcb.200705163/DC1).


Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.

Blower MD, Feric E, Weis K, Heald R - J. Cell Biol. (2007)

Specific mRNAs are localized to microtubules in mitotic human extracts. (A) Mitotic extracts were prepared from synchronized human cells, and microtubule asters induced by the addition of taxol were fixed and spun onto coverslips and stained for RNA (using SYTO RNASelect) and tubulin. RNA was localized to microtubule asters in a granular staining pattern. (B) Asters from this extract were pelleted through a glycerol cushion and isolated RNA was run on an agarose gel and stained with ethidium bromide. rRNA and mRNA pelleted more efficiently from extracts when microtubules were polymerized. Pelleted nucleic acid diminished upon treatment with RNaseA. (C) MT-mRNA and total mRNA were hybridized to Affymetrix microarrays as described in Fig. 1. As seen with X. laevis, specific mRNAs were enriched on mitotic human microtubules, whereas the vast majority of mRNAs were not. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2373496&req=5

fig2: Specific mRNAs are localized to microtubules in mitotic human extracts. (A) Mitotic extracts were prepared from synchronized human cells, and microtubule asters induced by the addition of taxol were fixed and spun onto coverslips and stained for RNA (using SYTO RNASelect) and tubulin. RNA was localized to microtubule asters in a granular staining pattern. (B) Asters from this extract were pelleted through a glycerol cushion and isolated RNA was run on an agarose gel and stained with ethidium bromide. rRNA and mRNA pelleted more efficiently from extracts when microtubules were polymerized. Pelleted nucleic acid diminished upon treatment with RNaseA. (C) MT-mRNA and total mRNA were hybridized to Affymetrix microarrays as described in Fig. 1. As seen with X. laevis, specific mRNAs were enriched on mitotic human microtubules, whereas the vast majority of mRNAs were not. Bars, 5 μm.
Mentions: We next asked whether localization of specific mRNAs to microtubules during mitosis is conserved among different cell types and species. Synchronized HeLa cells were used to prepare mitotic extracts that assemble radial microtubule asters when treated with taxol, reproducing many aspects of mitotic microtubule organization (Gaglio et al., 1995; Mack and Compton, 2001). RNA localized in a granular pattern on taxol microtubule asters in HeLa extracts, and both rRNA and mRNA specifically copurified with microtubules (Fig. 2, A and B). To identify MT-mRNAs in mitotic HeLa extracts, we hybridized MT-RNA– derived cRNAs to Affymetrix microarrays and compared the abundance of mRNAs in the microtubule sample to the total extract. Similar to our results with X. laevis, we observed that there was a continuum of mRNA binding to microtubules, with ∼10% of all mRNAs enriched 1.5-fold or more on mitotic microtubules (Fig. 2 C), and that mRNAs encoding proteins involved in various aspects of mitosis and DNA metabolism (replication, repair, and topology) were overrepresented in the MT-mRNA fraction (Table S5). The CPE was also overrepresented in the UTRs of MT-mRNAs with 5.7% of them possessing this element, which is a 4.1-fold enrichment compared with all mRNAs (Table S3, available at http://www.jcb.org/cgi/content/full/jcb.200705163/DC1).

Bottom Line: We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis.Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes.Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. blower@molbio.mgh.harvard.edu

ABSTRACT
RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.

Show MeSH