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Structural imprints in vivo decode RNA regulatory mechanisms.

Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY - Nature (2015)

Bottom Line: In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data.Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide.These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

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NAI-N3 is a novel RNA acylation reagent that enables RNA purificationa, Chemical schematic of RNA acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate. b, ATP acylation gel shift showing ATP acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate.
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Figure 7: NAI-N3 is a novel RNA acylation reagent that enables RNA purificationa, Chemical schematic of RNA acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate. b, ATP acylation gel shift showing ATP acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate.

Mentions: We designed, synthesized, and tested a novel bifunctional chemical probe for in vivo RNA structure profiling genome-wide (NAI-N3, Fig. 1a, 1b, and Extended Data Fig 1). NAI-N3 adds an azide group to NAI, a cell permeable SHAPE reagent5. By using copper-free click chemistry, a biotin moiety is selectively and efficiently added to NAI-N3-modified RNA, providing a stringent purification handle with streptavidin beads (Fig. 1c, Extended Data Fig. 2). NAI-N3 generated identical profiles of RT stops to those obtained using our previously designed SHAPE reagent5. The fidelity of structural measurements was not affected by “clicking” biotin onto the NAI-N3 nor by molecular crowding of proteins, and NAI-N3 showed uniform modification of all bases in denatured RNAs (Extended Data Fig. 3). We term this new chemoaffinity structure probing methodology In VivoClick SHAPE (icSHAPE); this method can also be applied to any ex vivo preparation of RNA with slight modifications.


Structural imprints in vivo decode RNA regulatory mechanisms.

Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY - Nature (2015)

NAI-N3 is a novel RNA acylation reagent that enables RNA purificationa, Chemical schematic of RNA acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate. b, ATP acylation gel shift showing ATP acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4376618&req=5

Figure 7: NAI-N3 is a novel RNA acylation reagent that enables RNA purificationa, Chemical schematic of RNA acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate. b, ATP acylation gel shift showing ATP acylation and copper-free ‘click’ chemistry utilizing NAI-N3 and dibenzocyclooxtyne-biotin conjugate.
Mentions: We designed, synthesized, and tested a novel bifunctional chemical probe for in vivo RNA structure profiling genome-wide (NAI-N3, Fig. 1a, 1b, and Extended Data Fig 1). NAI-N3 adds an azide group to NAI, a cell permeable SHAPE reagent5. By using copper-free click chemistry, a biotin moiety is selectively and efficiently added to NAI-N3-modified RNA, providing a stringent purification handle with streptavidin beads (Fig. 1c, Extended Data Fig. 2). NAI-N3 generated identical profiles of RT stops to those obtained using our previously designed SHAPE reagent5. The fidelity of structural measurements was not affected by “clicking” biotin onto the NAI-N3 nor by molecular crowding of proteins, and NAI-N3 showed uniform modification of all bases in denatured RNAs (Extended Data Fig. 3). We term this new chemoaffinity structure probing methodology In VivoClick SHAPE (icSHAPE); this method can also be applied to any ex vivo preparation of RNA with slight modifications.

Bottom Line: In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data.Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide.These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

Show MeSH