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Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Laver JD, Li X, Ancevicius K, Westwood JT, Smibert CA, Morris QD, Lipshitz HD - Nucleic Acids Res. (2013)

Bottom Line: We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos.First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts.These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8, Department of Cell & Systems Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 and Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

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Related in: MedlinePlus

Enrichment of expressed transcripts in Staufen RIPs using synthetic anti-Staufen and anti-GFP-Staufen antibodies. The average, across three biological replicates, of the log2 microarray signal intensities of each expressed transcript in the anti-Staufen and control RIPs were plotted against each other (A, C, E). Highlighted in red and shown at the individual replicate level in the adjacent boxplots (B, D, F) are the transcripts that were found, through Significance Analysis of Microarray two-class analysis, to be significantly enriched in the anti-Staufen versus the control RIPs. Those transcripts with an FDR < 5% and passing a fold enrichment cut-off of at least two in the synthetic anti-Staufen experiments are shown in panels (A) and (B), and those with an FDR < 5% and passing fold enrichment cut-offs of at least two and five in the transgenic anti-GFP experiments are shown in panels (C, D) and panels (E, F), respectively. The three previously identified targets of Staufen (bicoid, oskar and prospero) are labelled in each scatter plot and further denoted by crosses. In (A, C, E), solid diagonal lines represent no enrichment, and dotted diagonal lines represent 2-fold (A, C) or 5-fold (E) enrichment or depletion.
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gkt702-F1: Enrichment of expressed transcripts in Staufen RIPs using synthetic anti-Staufen and anti-GFP-Staufen antibodies. The average, across three biological replicates, of the log2 microarray signal intensities of each expressed transcript in the anti-Staufen and control RIPs were plotted against each other (A, C, E). Highlighted in red and shown at the individual replicate level in the adjacent boxplots (B, D, F) are the transcripts that were found, through Significance Analysis of Microarray two-class analysis, to be significantly enriched in the anti-Staufen versus the control RIPs. Those transcripts with an FDR < 5% and passing a fold enrichment cut-off of at least two in the synthetic anti-Staufen experiments are shown in panels (A) and (B), and those with an FDR < 5% and passing fold enrichment cut-offs of at least two and five in the transgenic anti-GFP experiments are shown in panels (C, D) and panels (E, F), respectively. The three previously identified targets of Staufen (bicoid, oskar and prospero) are labelled in each scatter plot and further denoted by crosses. In (A, C, E), solid diagonal lines represent no enrichment, and dotted diagonal lines represent 2-fold (A, C) or 5-fold (E) enrichment or depletion.

Mentions: To identify mRNAs associated with Staufen in early Drosophila embryos, we performed RIP-Chip using two complementary approaches. First, we carried out RIP-Chip of endogenous Staufen from wild-type 0–3 h old embryos using a synthetic antibody, anti-Staufen 2A5, that we previously showed immunoprecipitates Staufen protein along with bicoid mRNA (36). As a negative control, we performed immunoprecipitations using a control antibody (C1) derived from the same synthetic antibody library as anti-Staufen 2A5 (36). We identified 46 genes whose mRNAs were enriched at least 2-fold in Staufen immunoprecipitates compared with negative control immunoprecipitates and had an FDR of <5% (Figure 1A and B and Table 1; Supplementary Figures S1 and S2; Supplementary Table S1; see ‘Materials and Methods’ section for details). All three previously identified Staufen target mRNAs, bicoid, oskar and prospero, were among these 46, and bicoid mRNA was the most highly enriched target identified. Validation experiments using RT-qPCR are presented in Supplementary Table S2.Figure 1.


Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Laver JD, Li X, Ancevicius K, Westwood JT, Smibert CA, Morris QD, Lipshitz HD - Nucleic Acids Res. (2013)

Enrichment of expressed transcripts in Staufen RIPs using synthetic anti-Staufen and anti-GFP-Staufen antibodies. The average, across three biological replicates, of the log2 microarray signal intensities of each expressed transcript in the anti-Staufen and control RIPs were plotted against each other (A, C, E). Highlighted in red and shown at the individual replicate level in the adjacent boxplots (B, D, F) are the transcripts that were found, through Significance Analysis of Microarray two-class analysis, to be significantly enriched in the anti-Staufen versus the control RIPs. Those transcripts with an FDR < 5% and passing a fold enrichment cut-off of at least two in the synthetic anti-Staufen experiments are shown in panels (A) and (B), and those with an FDR < 5% and passing fold enrichment cut-offs of at least two and five in the transgenic anti-GFP experiments are shown in panels (C, D) and panels (E, F), respectively. The three previously identified targets of Staufen (bicoid, oskar and prospero) are labelled in each scatter plot and further denoted by crosses. In (A, C, E), solid diagonal lines represent no enrichment, and dotted diagonal lines represent 2-fold (A, C) or 5-fold (E) enrichment or depletion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814352&req=5

gkt702-F1: Enrichment of expressed transcripts in Staufen RIPs using synthetic anti-Staufen and anti-GFP-Staufen antibodies. The average, across three biological replicates, of the log2 microarray signal intensities of each expressed transcript in the anti-Staufen and control RIPs were plotted against each other (A, C, E). Highlighted in red and shown at the individual replicate level in the adjacent boxplots (B, D, F) are the transcripts that were found, through Significance Analysis of Microarray two-class analysis, to be significantly enriched in the anti-Staufen versus the control RIPs. Those transcripts with an FDR < 5% and passing a fold enrichment cut-off of at least two in the synthetic anti-Staufen experiments are shown in panels (A) and (B), and those with an FDR < 5% and passing fold enrichment cut-offs of at least two and five in the transgenic anti-GFP experiments are shown in panels (C, D) and panels (E, F), respectively. The three previously identified targets of Staufen (bicoid, oskar and prospero) are labelled in each scatter plot and further denoted by crosses. In (A, C, E), solid diagonal lines represent no enrichment, and dotted diagonal lines represent 2-fold (A, C) or 5-fold (E) enrichment or depletion.
Mentions: To identify mRNAs associated with Staufen in early Drosophila embryos, we performed RIP-Chip using two complementary approaches. First, we carried out RIP-Chip of endogenous Staufen from wild-type 0–3 h old embryos using a synthetic antibody, anti-Staufen 2A5, that we previously showed immunoprecipitates Staufen protein along with bicoid mRNA (36). As a negative control, we performed immunoprecipitations using a control antibody (C1) derived from the same synthetic antibody library as anti-Staufen 2A5 (36). We identified 46 genes whose mRNAs were enriched at least 2-fold in Staufen immunoprecipitates compared with negative control immunoprecipitates and had an FDR of <5% (Figure 1A and B and Table 1; Supplementary Figures S1 and S2; Supplementary Table S1; see ‘Materials and Methods’ section for details). All three previously identified Staufen target mRNAs, bicoid, oskar and prospero, were among these 46, and bicoid mRNA was the most highly enriched target identified. Validation experiments using RT-qPCR are presented in Supplementary Table S2.Figure 1.

Bottom Line: We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos.First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts.These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8, Department of Cell & Systems Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 and Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Show MeSH
Related in: MedlinePlus