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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.

Show MeSH
Predicted SRSs map with high precision to the known Staufen-binding regions in Drosophila bicoid and human ARF1 3′UTRs. Mapping of the predicted Drosophila SRSs to experimentally determined in vivo Staufen-binding regions in (A) Drosophila bicoid (12) and (B) human ARF1 (31) 3′UTRs. The grey shading in the background indicates the regions that are important for Staufen binding in vivo as defined in those studies. The x-axis presents the relevant region of the 3′UTR in nucleotides, starting from the first nucleotide after the stop codon. The black lines represent the entire span of each predicted SRS hit that is indicated on the y-axis, mapped onto the 3′UTR sequence. For each SRS, the 5′-most nucleotide in the corresponding 15 of 19 or 10 of 12 motif is connected to the paired nucleotide in the partner arm by a line.
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gkt702-F10: Predicted SRSs map with high precision to the known Staufen-binding regions in Drosophila bicoid and human ARF1 3′UTRs. Mapping of the predicted Drosophila SRSs to experimentally determined in vivo Staufen-binding regions in (A) Drosophila bicoid (12) and (B) human ARF1 (31) 3′UTRs. The grey shading in the background indicates the regions that are important for Staufen binding in vivo as defined in those studies. The x-axis presents the relevant region of the 3′UTR in nucleotides, starting from the first nucleotide after the stop codon. The black lines represent the entire span of each predicted SRS hit that is indicated on the y-axis, mapped onto the 3′UTR sequence. For each SRS, the 5′-most nucleotide in the corresponding 15 of 19 or 10 of 12 motif is connected to the paired nucleotide in the partner arm by a line.

Mentions: Finally, to assess whether SRSs map to the experimentally determined in vivo Staufen-binding sites, we focused on Drosophila bicoid (13,31) and human ARF1 (32). In the bicoid 3′UTR, the SRSs mapped almost exclusively to the three experimentally determined Staufen-binding regions (Figure 10A): for Type II SRSs, the precision was 1.0, and for Type III SRSs, the precision was 0.94 (baseline precision = 0.47; there were no Type I SRSs). For human ARF1’s 3′UTR, again, the SRSs mapped almost exclusively to the two known Staufen1-binding regions (Figure 10B): for all three types of SRSs, the precision was 1.0 (baseline precision = 0.13). Taken together, these results provide strong evidence that the computationally identified structures correspond to bona fide, in vivo Staufen-binding sites.Figure 10.


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)

Predicted SRSs map with high precision to the known Staufen-binding regions in Drosophila bicoid and human ARF1 3′UTRs. Mapping of the predicted Drosophila SRSs to experimentally determined in vivo Staufen-binding regions in (A) Drosophila bicoid (12) and (B) human ARF1 (31) 3′UTRs. The grey shading in the background indicates the regions that are important for Staufen binding in vivo as defined in those studies. The x-axis presents the relevant region of the 3′UTR in nucleotides, starting from the first nucleotide after the stop codon. The black lines represent the entire span of each predicted SRS hit that is indicated on the y-axis, mapped onto the 3′UTR sequence. For each SRS, the 5′-most nucleotide in the corresponding 15 of 19 or 10 of 12 motif is connected to the paired nucleotide in the partner arm by a line.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt702-F10: Predicted SRSs map with high precision to the known Staufen-binding regions in Drosophila bicoid and human ARF1 3′UTRs. Mapping of the predicted Drosophila SRSs to experimentally determined in vivo Staufen-binding regions in (A) Drosophila bicoid (12) and (B) human ARF1 (31) 3′UTRs. The grey shading in the background indicates the regions that are important for Staufen binding in vivo as defined in those studies. The x-axis presents the relevant region of the 3′UTR in nucleotides, starting from the first nucleotide after the stop codon. The black lines represent the entire span of each predicted SRS hit that is indicated on the y-axis, mapped onto the 3′UTR sequence. For each SRS, the 5′-most nucleotide in the corresponding 15 of 19 or 10 of 12 motif is connected to the paired nucleotide in the partner arm by a line.
Mentions: Finally, to assess whether SRSs map to the experimentally determined in vivo Staufen-binding sites, we focused on Drosophila bicoid (13,31) and human ARF1 (32). In the bicoid 3′UTR, the SRSs mapped almost exclusively to the three experimentally determined Staufen-binding regions (Figure 10A): for Type II SRSs, the precision was 1.0, and for Type III SRSs, the precision was 0.94 (baseline precision = 0.47; there were no Type I SRSs). For human ARF1’s 3′UTR, again, the SRSs mapped almost exclusively to the two known Staufen1-binding regions (Figure 10B): for all three types of SRSs, the precision was 1.0 (baseline precision = 0.13). Taken together, these results provide strong evidence that the computationally identified structures correspond to bona fide, in vivo Staufen-binding sites.Figure 10.

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