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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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Characteristics of the stems bound by Staufen. We compared the 10 of 12 (A) and 15 of 19 (B) structures in the Staufen targets versus non-targets, by characterizing the structural features: (i) number of mismatches; (ii) number of unpaired bases; (iii) number of bulges; (iv) number of internal loops. If there was more than one structure in a transcript’s 3′UTR, the feature with the minimal score was used to represent the gene. The Wilcoxon rank sum test was used to assess the feature in Staufen targets (the solid line) versus non-targets (the dashed line). pos: targets; neg: non-targets.
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gkt702-F7: Characteristics of the stems bound by Staufen. We compared the 10 of 12 (A) and 15 of 19 (B) structures in the Staufen targets versus non-targets, by characterizing the structural features: (i) number of mismatches; (ii) number of unpaired bases; (iii) number of bulges; (iv) number of internal loops. If there was more than one structure in a transcript’s 3′UTR, the feature with the minimal score was used to represent the gene. The Wilcoxon rank sum test was used to assess the feature in Staufen targets (the solid line) versus non-targets (the dashed line). pos: targets; neg: non-targets.

Mentions: Many transcripts in both the target and non-target sets contain multiple [12,10] and [19,15] structures; as such, we next considered the same criteria at the transcript level to determine features associated with the ‘best’ Staufen site in each transcript. We did this by assigning each transcript a mismatch, unpaired base, and loop count as well as loop length equal to the minimum of those values across all [12,10] and [19,15] structures in that transcript’s 3′UTR. This analysis also revealed a significantly lower number of unpaired bases in the ‘best’ structure in Staufen target transcripts compared with non-targets, which also manifested as an even stronger preference for balanced stems (i.e. those containing only paired or mismatched bases and no unpaired bases) (Figure 7). In particular, 82% of Staufen targets that had at least one [12,10] structure contained a balanced [12,10] structure (compared with 47% in non-targets). We use the designation [12,10,0] to describe these balanced structures where the last number refers to the maximum number of unpaired bases in the structure. The enrichment among the Staufen targets for transcripts containing balanced [19,15] structures (i.e. [19,15,0]) was even more striking (67 versus 12%). Because many more Staufen target transcripts have [19,15] structures than do non-targets, if we define a Staufen-bound transcript as one containing a [19,15] that is balanced (i.e. [19,15,0]), this rule would be satisfied by 44% (n = 24) of the target transcripts but only 3% of the non-target transcripts (n = 42).Figure 7.


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)

Characteristics of the stems bound by Staufen. We compared the 10 of 12 (A) and 15 of 19 (B) structures in the Staufen targets versus non-targets, by characterizing the structural features: (i) number of mismatches; (ii) number of unpaired bases; (iii) number of bulges; (iv) number of internal loops. If there was more than one structure in a transcript’s 3′UTR, the feature with the minimal score was used to represent the gene. The Wilcoxon rank sum test was used to assess the feature in Staufen targets (the solid line) versus non-targets (the dashed line). pos: targets; neg: non-targets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt702-F7: Characteristics of the stems bound by Staufen. We compared the 10 of 12 (A) and 15 of 19 (B) structures in the Staufen targets versus non-targets, by characterizing the structural features: (i) number of mismatches; (ii) number of unpaired bases; (iii) number of bulges; (iv) number of internal loops. If there was more than one structure in a transcript’s 3′UTR, the feature with the minimal score was used to represent the gene. The Wilcoxon rank sum test was used to assess the feature in Staufen targets (the solid line) versus non-targets (the dashed line). pos: targets; neg: non-targets.
Mentions: Many transcripts in both the target and non-target sets contain multiple [12,10] and [19,15] structures; as such, we next considered the same criteria at the transcript level to determine features associated with the ‘best’ Staufen site in each transcript. We did this by assigning each transcript a mismatch, unpaired base, and loop count as well as loop length equal to the minimum of those values across all [12,10] and [19,15] structures in that transcript’s 3′UTR. This analysis also revealed a significantly lower number of unpaired bases in the ‘best’ structure in Staufen target transcripts compared with non-targets, which also manifested as an even stronger preference for balanced stems (i.e. those containing only paired or mismatched bases and no unpaired bases) (Figure 7). In particular, 82% of Staufen targets that had at least one [12,10] structure contained a balanced [12,10] structure (compared with 47% in non-targets). We use the designation [12,10,0] to describe these balanced structures where the last number refers to the maximum number of unpaired bases in the structure. The enrichment among the Staufen targets for transcripts containing balanced [19,15] structures (i.e. [19,15,0]) was even more striking (67 versus 12%). Because many more Staufen target transcripts have [19,15] structures than do non-targets, if we define a Staufen-bound transcript as one containing a [19,15] that is balanced (i.e. [19,15,0]), this rule would be satisfied by 44% (n = 24) of the target transcripts but only 3% of the non-target transcripts (n = 42).Figure 7.

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