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Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome.

Riordan DP, Herschlag D, Brown PO - Nucleic Acids Res. (2010)

Bottom Line: We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements.Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3.Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA. driordan@stanford.edu

ABSTRACT
Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA-protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

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RNA recognition elements determined by in vitro selection. RNA motifs determined by analysis of SELEX clone sequences are depicted for each RBP. The −log10 P-values of the significance of motif enrichment in clones from the two distinct SELEX libraries (‘L1’ and ‘L2’) are represented in a red color scale. For each motif, the −log10 P-value of the significance of genome-wide enrichment for motif sites in segments of its mRNA targets bound in vivo is also color-coded. Motifs are listed in the order they are discussed in the text. The gray box indicates data are not available because all L2 clones inadvertently contain an Hrb1 motif site in their 3′ constant region. Asterisks denote motifs that correspond to previously reported binding sites for the associated RBP. For exact data values and details see Supplementary Data S3.
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Figure 3: RNA recognition elements determined by in vitro selection. RNA motifs determined by analysis of SELEX clone sequences are depicted for each RBP. The −log10 P-values of the significance of motif enrichment in clones from the two distinct SELEX libraries (‘L1’ and ‘L2’) are represented in a red color scale. For each motif, the −log10 P-value of the significance of genome-wide enrichment for motif sites in segments of its mRNA targets bound in vivo is also color-coded. Motifs are listed in the order they are discussed in the text. The gray box indicates data are not available because all L2 clones inadvertently contain an Hrb1 motif site in their 3′ constant region. Asterisks denote motifs that correspond to previously reported binding sites for the associated RBP. For exact data values and details see Supplementary Data S3.

Mentions: We developed an efficient SELEX (systematic evolution of ligands by exponential enrichment) (19,20) protocol for selecting RNA ligands that specifically bind to individual RBPs. The approach takes advantage of the yeast genome-wide collection of TAP-tagged strains (21). Binding reactions were performed by adding in vitro-transcribed RNA pools, consisting of 30 randomized bases flanked by two 20 base constant regions, to a cell lysate containing the TAP-tagged RBP of interest. Each reaction contained ∼6 × 1013 molecules of library RNA, which theoretically represents ∼600-fold coverage of all 20-mers in the randomized pool. In each round of selection, the RBP and its associated RNAs were selectively isolated, the recovered RNAs were reverse transcribed and the products were amplified by PCR using primers specific to the flanking constant sequences. Four cycles of selection were performed for each RBP. At each cycle, the fraction of input library selectively bound by the RBP was monitored by qPCR. All selections were performed using two distinct libraries (L1 and L2) with different constant sequences to facilitate the detection of library-specific features that could have contributed to the selection. For 10 of the 12 RBPs tested (excepting Bfr1 and Khd1), serial enrichment yielded RNA pools with apparent affinity significantly above the background level in the unselected pool for both libraries (Supplementary Data S3). We inferred RNA recognition elements by manually analyzing the sequences of individual molecules enriched in each selection, then checking for enrichment of the inferred motifs in the empirically derived in vivo RNA targets of the corresponding RBP (Figure 3).Figure 3.


Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome.

Riordan DP, Herschlag D, Brown PO - Nucleic Acids Res. (2010)

RNA recognition elements determined by in vitro selection. RNA motifs determined by analysis of SELEX clone sequences are depicted for each RBP. The −log10 P-values of the significance of motif enrichment in clones from the two distinct SELEX libraries (‘L1’ and ‘L2’) are represented in a red color scale. For each motif, the −log10 P-value of the significance of genome-wide enrichment for motif sites in segments of its mRNA targets bound in vivo is also color-coded. Motifs are listed in the order they are discussed in the text. The gray box indicates data are not available because all L2 clones inadvertently contain an Hrb1 motif site in their 3′ constant region. Asterisks denote motifs that correspond to previously reported binding sites for the associated RBP. For exact data values and details see Supplementary Data S3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045596&req=5

Figure 3: RNA recognition elements determined by in vitro selection. RNA motifs determined by analysis of SELEX clone sequences are depicted for each RBP. The −log10 P-values of the significance of motif enrichment in clones from the two distinct SELEX libraries (‘L1’ and ‘L2’) are represented in a red color scale. For each motif, the −log10 P-value of the significance of genome-wide enrichment for motif sites in segments of its mRNA targets bound in vivo is also color-coded. Motifs are listed in the order they are discussed in the text. The gray box indicates data are not available because all L2 clones inadvertently contain an Hrb1 motif site in their 3′ constant region. Asterisks denote motifs that correspond to previously reported binding sites for the associated RBP. For exact data values and details see Supplementary Data S3.
Mentions: We developed an efficient SELEX (systematic evolution of ligands by exponential enrichment) (19,20) protocol for selecting RNA ligands that specifically bind to individual RBPs. The approach takes advantage of the yeast genome-wide collection of TAP-tagged strains (21). Binding reactions were performed by adding in vitro-transcribed RNA pools, consisting of 30 randomized bases flanked by two 20 base constant regions, to a cell lysate containing the TAP-tagged RBP of interest. Each reaction contained ∼6 × 1013 molecules of library RNA, which theoretically represents ∼600-fold coverage of all 20-mers in the randomized pool. In each round of selection, the RBP and its associated RNAs were selectively isolated, the recovered RNAs were reverse transcribed and the products were amplified by PCR using primers specific to the flanking constant sequences. Four cycles of selection were performed for each RBP. At each cycle, the fraction of input library selectively bound by the RBP was monitored by qPCR. All selections were performed using two distinct libraries (L1 and L2) with different constant sequences to facilitate the detection of library-specific features that could have contributed to the selection. For 10 of the 12 RBPs tested (excepting Bfr1 and Khd1), serial enrichment yielded RNA pools with apparent affinity significantly above the background level in the unselected pool for both libraries (Supplementary Data S3). We inferred RNA recognition elements by manually analyzing the sequences of individual molecules enriched in each selection, then checking for enrichment of the inferred motifs in the empirically derived in vivo RNA targets of the corresponding RBP (Figure 3).Figure 3.

Bottom Line: We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements.Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3.Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA. driordan@stanford.edu

ABSTRACT
Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA-protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

Show MeSH
Related in: MedlinePlus