RNA SHAPE analysis in living cells - PubMed (original) (raw)

RNA SHAPE analysis in living cells

Robert C Spitale et al. Nat Chem Biol. 2013 Jan.

Abstract

RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

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Conflict of interest statement

Competing Financial Interests:

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Design and experimental evaluation of NAI and FAI

(a) Structural comparison between NMIA, FAI, and NAI. (b) Chemical mechanism of 2’-O-acylation of RNA. (c) Time course of ATP modification by NAI and FAI. (d) Correlation of 2’-RNA modification of 5S rRNA in vitro. (e) Denaturing gel electrophoresis of NAI modification of 5S rRNA in cells. (f) Secondary structure mapping of NAI modification of 5S rRNA in cells. (g) Three-dimensional model of S.C. 5SrRNA with modifications superimposed onto the structure (PDB 3U5H).

Figure 2

Figure 2. 5S rRNA has different modification patterns in cells

(a) Denaturing gel electrophoretic analysis of NAI modification of 5S rRNA in M. musculus Embyronic Stem cells and in vitro. (b) Normalized Differential profile of M. musculus Embyronic Stem cell 5S rRNA. (c) Three-dimensional model of S.C. 5SrRNA with differential modifications superimposed onto the structure (PDB 3U5H). (d) Closeup view of hyperreactivity of M.M. A49 and S.C. U50. U50 is near the kink of helix II and helix III, This conformation, wich allows Loop C to interact with 28S rRNA promotes the ejection of S.C. U50 2’-OH thus rendering it more dynamic and reactive to NAI modification. (e) Zoomed-in view of a three-nucleotide bridge that joins loop A to helix II. This conformation results in docking of A11, hiding its 2’-OH through a hydrogen bond with A13-OP2. In addition C10 is forced into a stacking interaction with PHE20, which exposes the 2’-OH, potentially increasing its reactivity with NAI. (f). Close up view of differential modification of Helix IV. The ribosome crystal structure shows many of the residues that are hypomodified in cells are engaged in extensive interactions with ribosomal RNA. Shown is the interaction of U86 with 28S rRNA. Such extensive bonding and stacking may be stabilizing the internucleotide linkages of these residues, limiting their acylation reactivity. (g) Three-dimensional model of S.C. 5SrRNA colored by b-factor (PDB 3U5H). (h) Denaturing gel electrophoretic analysis of NAI modification of 5S rRNA in S. cerevisiae cells and in vitro. (i) Normalized differential profile of S. cerevisiae 5S rRNA. Residues are labeled for their importance in 5S rRNA function as noted in reference 31.

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