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Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

Jambor H, Surendranath V, Kalinka AT, Mejstrik P, Saalfeld S, Tomancak P - Elife (2015)

Bottom Line: We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte.Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs.Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

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Posterior mRNA localization is impaired in posterior localization pathway mutants.(A) Localization of the novel posterior candidate mRNAs vkg, TwdlG, PI3K21B, and zpg is lost in egg-chambers that prematurely depolymerize the microtubules (flies homozygous for SpireRP), are mutant for the RNA binding protein Staufen (flies homozygous for StauD3) or mutant for the EJC protein Barentz (flies homozygous for Btz1). The candidate mRNAs are mis-localized in a manner similar to oskar mRNA. In Btz1 egg-chambers a weak enrichment of vkg mRNA remained that in rare instances is also observed for oskar mRNA. (B) In egg-chambers either lacking functional Oskar protein or posterior oskar mRNA, the localization of the candidate posterior mRNAs was lost: In Oskar protein mutant egg-chambers (osk84/Df(3R)pXT103), oskar mRNA is initially localized at stage 9 but successively detaches from the posterior pole resulting in reduced oskar mRNA at the posterior pole from stage 10 onwards. A similar reduction from stage 9 to 10 was seen for vkg and TwdlG mRNAs, while PI3K21B (already in wild type being localized at low levels) and zpg mRNA (in wild type localized after stage 9) never showed localization. In egg-chambers that do not express posterior oskar mRNA (oskar 3′UTR/+; oskA87/Df(3R)pXT103; Jenny et al., 2006) none of the candidate posterior mRNAs localized. (Expression of the non-localizing oskar 3′UTR is necessary to rescue the early oogenesis arrest.) (A–B) FISH experiments showing the RNA in green and DNA (labelled with DAPI) in magenta. Scale bars 30 μm.DOI:http://dx.doi.org/10.7554/eLife.05003.013
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fig3s5: Posterior mRNA localization is impaired in posterior localization pathway mutants.(A) Localization of the novel posterior candidate mRNAs vkg, TwdlG, PI3K21B, and zpg is lost in egg-chambers that prematurely depolymerize the microtubules (flies homozygous for SpireRP), are mutant for the RNA binding protein Staufen (flies homozygous for StauD3) or mutant for the EJC protein Barentz (flies homozygous for Btz1). The candidate mRNAs are mis-localized in a manner similar to oskar mRNA. In Btz1 egg-chambers a weak enrichment of vkg mRNA remained that in rare instances is also observed for oskar mRNA. (B) In egg-chambers either lacking functional Oskar protein or posterior oskar mRNA, the localization of the candidate posterior mRNAs was lost: In Oskar protein mutant egg-chambers (osk84/Df(3R)pXT103), oskar mRNA is initially localized at stage 9 but successively detaches from the posterior pole resulting in reduced oskar mRNA at the posterior pole from stage 10 onwards. A similar reduction from stage 9 to 10 was seen for vkg and TwdlG mRNAs, while PI3K21B (already in wild type being localized at low levels) and zpg mRNA (in wild type localized after stage 9) never showed localization. In egg-chambers that do not express posterior oskar mRNA (oskar 3′UTR/+; oskA87/Df(3R)pXT103; Jenny et al., 2006) none of the candidate posterior mRNAs localized. (Expression of the non-localizing oskar 3′UTR is necessary to rescue the early oogenesis arrest.) (A–B) FISH experiments showing the RNA in green and DNA (labelled with DAPI) in magenta. Scale bars 30 μm.DOI:http://dx.doi.org/10.7554/eLife.05003.013

Mentions: However, mRNA localization requires more than an intact microtubule cytoskeleton. We therefore next investigated the localization of candidate posterior mRNAs in mutant egg-chambers that affect the localization of the known posterior mRNA, oskar. Posterior transport of oskar mRNA requires components of the exon junction complex, the RNA binding protein Staufen and an intact microtubule cytoskeleton (van Eeden et al., 2001; St Johnston et al., 1989; Ephrussi et al., 1991; Hachet and Ephrussi, 2001, 2004; Micklem et al., 2000). The posterior enrichment of the selected candidate mRNAs was severely reduced in egg-chambers mutant for an exon junction complex component (Btz1), that has a disrupted cytoskeleton (SpireRP) or that lack Staufen (StauD3) protein. The localization of all candidate posterior mRNAs resembled the mis-localized oskar mRNA in these mutant conditions (Figure 3—figure supplement 5A).


Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

Jambor H, Surendranath V, Kalinka AT, Mejstrik P, Saalfeld S, Tomancak P - Elife (2015)

Posterior mRNA localization is impaired in posterior localization pathway mutants.(A) Localization of the novel posterior candidate mRNAs vkg, TwdlG, PI3K21B, and zpg is lost in egg-chambers that prematurely depolymerize the microtubules (flies homozygous for SpireRP), are mutant for the RNA binding protein Staufen (flies homozygous for StauD3) or mutant for the EJC protein Barentz (flies homozygous for Btz1). The candidate mRNAs are mis-localized in a manner similar to oskar mRNA. In Btz1 egg-chambers a weak enrichment of vkg mRNA remained that in rare instances is also observed for oskar mRNA. (B) In egg-chambers either lacking functional Oskar protein or posterior oskar mRNA, the localization of the candidate posterior mRNAs was lost: In Oskar protein mutant egg-chambers (osk84/Df(3R)pXT103), oskar mRNA is initially localized at stage 9 but successively detaches from the posterior pole resulting in reduced oskar mRNA at the posterior pole from stage 10 onwards. A similar reduction from stage 9 to 10 was seen for vkg and TwdlG mRNAs, while PI3K21B (already in wild type being localized at low levels) and zpg mRNA (in wild type localized after stage 9) never showed localization. In egg-chambers that do not express posterior oskar mRNA (oskar 3′UTR/+; oskA87/Df(3R)pXT103; Jenny et al., 2006) none of the candidate posterior mRNAs localized. (Expression of the non-localizing oskar 3′UTR is necessary to rescue the early oogenesis arrest.) (A–B) FISH experiments showing the RNA in green and DNA (labelled with DAPI) in magenta. Scale bars 30 μm.DOI:http://dx.doi.org/10.7554/eLife.05003.013
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Related In: Results  -  Collection

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fig3s5: Posterior mRNA localization is impaired in posterior localization pathway mutants.(A) Localization of the novel posterior candidate mRNAs vkg, TwdlG, PI3K21B, and zpg is lost in egg-chambers that prematurely depolymerize the microtubules (flies homozygous for SpireRP), are mutant for the RNA binding protein Staufen (flies homozygous for StauD3) or mutant for the EJC protein Barentz (flies homozygous for Btz1). The candidate mRNAs are mis-localized in a manner similar to oskar mRNA. In Btz1 egg-chambers a weak enrichment of vkg mRNA remained that in rare instances is also observed for oskar mRNA. (B) In egg-chambers either lacking functional Oskar protein or posterior oskar mRNA, the localization of the candidate posterior mRNAs was lost: In Oskar protein mutant egg-chambers (osk84/Df(3R)pXT103), oskar mRNA is initially localized at stage 9 but successively detaches from the posterior pole resulting in reduced oskar mRNA at the posterior pole from stage 10 onwards. A similar reduction from stage 9 to 10 was seen for vkg and TwdlG mRNAs, while PI3K21B (already in wild type being localized at low levels) and zpg mRNA (in wild type localized after stage 9) never showed localization. In egg-chambers that do not express posterior oskar mRNA (oskar 3′UTR/+; oskA87/Df(3R)pXT103; Jenny et al., 2006) none of the candidate posterior mRNAs localized. (Expression of the non-localizing oskar 3′UTR is necessary to rescue the early oogenesis arrest.) (A–B) FISH experiments showing the RNA in green and DNA (labelled with DAPI) in magenta. Scale bars 30 μm.DOI:http://dx.doi.org/10.7554/eLife.05003.013
Mentions: However, mRNA localization requires more than an intact microtubule cytoskeleton. We therefore next investigated the localization of candidate posterior mRNAs in mutant egg-chambers that affect the localization of the known posterior mRNA, oskar. Posterior transport of oskar mRNA requires components of the exon junction complex, the RNA binding protein Staufen and an intact microtubule cytoskeleton (van Eeden et al., 2001; St Johnston et al., 1989; Ephrussi et al., 1991; Hachet and Ephrussi, 2001, 2004; Micklem et al., 2000). The posterior enrichment of the selected candidate mRNAs was severely reduced in egg-chambers mutant for an exon junction complex component (Btz1), that has a disrupted cytoskeleton (SpireRP) or that lack Staufen (StauD3) protein. The localization of all candidate posterior mRNAs resembled the mis-localized oskar mRNA in these mutant conditions (Figure 3—figure supplement 5A).

Bottom Line: We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte.Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs.Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

Show MeSH
Related in: MedlinePlus