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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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Comparison of the synthetic anti-Staufen and anti-GFP-Staufen RIPs. (A) A Venn diagram [generated using the BioVenn web application (48)] shows overlap of Staufen targets from the synthetic anti-Staufen RIP with a fold enrichment cut-off of at least two (dark grey) and the transgenic anti-GFP RIPs with fold enrichment cut-offs of at least two (medium grey) or five (light grey). (B) The 503 genes from the anti-GFP 2-fold list were ranked according to decreasing fold enrichment and the 41 overlapping genes from the synthetic anti-Staufen 2-fold list were then overlaid in black showing that they represent genes with some of the highest fold-enrichments. (C) RT-qPCR analysis of the enrichment of the target mRNA bicoid and the reference mRNA RpL32 in Staufen RIPs conducted using wild-type extract and synthetic anti-Staufen, transgenic GFP-Staufen extract and synthetic anti-Staufen, and transgenic GFP-Staufen extract and anti-GFP. Each bar represents the average fold enrichment of the respective transcript in the anti-Staufen RIPs relative to the appropriate control. Error bars represent the standard error of the mean for n = 3 biological replicates.
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gkt702-F2: Comparison of the synthetic anti-Staufen and anti-GFP-Staufen RIPs. (A) A Venn diagram [generated using the BioVenn web application (48)] shows overlap of Staufen targets from the synthetic anti-Staufen RIP with a fold enrichment cut-off of at least two (dark grey) and the transgenic anti-GFP RIPs with fold enrichment cut-offs of at least two (medium grey) or five (light grey). (B) The 503 genes from the anti-GFP 2-fold list were ranked according to decreasing fold enrichment and the 41 overlapping genes from the synthetic anti-Staufen 2-fold list were then overlaid in black showing that they represent genes with some of the highest fold-enrichments. (C) RT-qPCR analysis of the enrichment of the target mRNA bicoid and the reference mRNA RpL32 in Staufen RIPs conducted using wild-type extract and synthetic anti-Staufen, transgenic GFP-Staufen extract and synthetic anti-Staufen, and transgenic GFP-Staufen extract and anti-GFP. Each bar represents the average fold enrichment of the respective transcript in the anti-Staufen RIPs relative to the appropriate control. Error bars represent the standard error of the mean for n = 3 biological replicates.

Mentions: For RIPs with synthetic anti-Staufen antibody, synthetic antibodies were expressed and purified as Fabs, and immunoprecipitations were as described (36) with only minor modifications. For anti-GFP-Staufen immunoprecipitations for western blots, RIP-Chip and quantitative PCR (qPCR) validation experiments, protein G magnetic beads (Invitrogen Cat. # 10004D) were first blocked (38), and immunoprecipitations were then performed using a protocol adapted from Invitrogen’s Dynabeads® Protein G protocol and Roche’s immunoprecipitation protocol for anti-GFP (Roche Cat. # 11814460001). The RNA retrieved from these immunoprecipitations was isolated using TRIzol (Invitrogen) following the manufacturer’s protocol and concentrated using RNA clean and concentrator 5 columns (Zymo Research Cat #R1015). For the comparison of synthetic antibody to anti-GFP RIPs (Figure 2C), a slightly different protocol was used, with the anti-GFP RIP protocol modified to be as similar as possible to the synthetic antibody RIP protocol. Details can be found in the Supplementary Materials and Methods.


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)

Comparison of the synthetic anti-Staufen and anti-GFP-Staufen RIPs. (A) A Venn diagram [generated using the BioVenn web application (48)] shows overlap of Staufen targets from the synthetic anti-Staufen RIP with a fold enrichment cut-off of at least two (dark grey) and the transgenic anti-GFP RIPs with fold enrichment cut-offs of at least two (medium grey) or five (light grey). (B) The 503 genes from the anti-GFP 2-fold list were ranked according to decreasing fold enrichment and the 41 overlapping genes from the synthetic anti-Staufen 2-fold list were then overlaid in black showing that they represent genes with some of the highest fold-enrichments. (C) RT-qPCR analysis of the enrichment of the target mRNA bicoid and the reference mRNA RpL32 in Staufen RIPs conducted using wild-type extract and synthetic anti-Staufen, transgenic GFP-Staufen extract and synthetic anti-Staufen, and transgenic GFP-Staufen extract and anti-GFP. Each bar represents the average fold enrichment of the respective transcript in the anti-Staufen RIPs relative to the appropriate control. Error bars represent the standard error of the mean for n = 3 biological replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt702-F2: Comparison of the synthetic anti-Staufen and anti-GFP-Staufen RIPs. (A) A Venn diagram [generated using the BioVenn web application (48)] shows overlap of Staufen targets from the synthetic anti-Staufen RIP with a fold enrichment cut-off of at least two (dark grey) and the transgenic anti-GFP RIPs with fold enrichment cut-offs of at least two (medium grey) or five (light grey). (B) The 503 genes from the anti-GFP 2-fold list were ranked according to decreasing fold enrichment and the 41 overlapping genes from the synthetic anti-Staufen 2-fold list were then overlaid in black showing that they represent genes with some of the highest fold-enrichments. (C) RT-qPCR analysis of the enrichment of the target mRNA bicoid and the reference mRNA RpL32 in Staufen RIPs conducted using wild-type extract and synthetic anti-Staufen, transgenic GFP-Staufen extract and synthetic anti-Staufen, and transgenic GFP-Staufen extract and anti-GFP. Each bar represents the average fold enrichment of the respective transcript in the anti-Staufen RIPs relative to the appropriate control. Error bars represent the standard error of the mean for n = 3 biological replicates.
Mentions: For RIPs with synthetic anti-Staufen antibody, synthetic antibodies were expressed and purified as Fabs, and immunoprecipitations were as described (36) with only minor modifications. For anti-GFP-Staufen immunoprecipitations for western blots, RIP-Chip and quantitative PCR (qPCR) validation experiments, protein G magnetic beads (Invitrogen Cat. # 10004D) were first blocked (38), and immunoprecipitations were then performed using a protocol adapted from Invitrogen’s Dynabeads® Protein G protocol and Roche’s immunoprecipitation protocol for anti-GFP (Roche Cat. # 11814460001). The RNA retrieved from these immunoprecipitations was isolated using TRIzol (Invitrogen) following the manufacturer’s protocol and concentrated using RNA clean and concentrator 5 columns (Zymo Research Cat #R1015). For the comparison of synthetic antibody to anti-GFP RIPs (Figure 2C), a slightly different protocol was used, with the anti-GFP RIP protocol modified to be as similar as possible to the synthetic antibody RIP protocol. Details can be found in the Supplementary Materials and Methods.

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