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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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icSHAPE Reveals Structural Profiles Associated with Translationa, Cartoon representation of ribosomes translating an mRNA. The uORF initiation site is represented by ribosome initiation upstream of the canonical start codon. The canonical start position is demarcated by “AUG”. The N-terminal truncation is represented as a ribosome initiating to the 3’-end of the canonical start “AUG”. b, icSHAPE profile at canonical start codon position. c, icSHAPE profiles at uORF and N-terminal truncation sites. d, Cartoon representation of a paused ribosome and its corresponding A-P-E sites. A: acceptor; P: peptidyl-transferase; E: exit. e, The icSHAPE profile at ribosome pause sites. f, icSHAPE profile at negative control sites for pause sequences. Gray box highlights a region of structural difference upstream of true pause sites vs. controls.
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Figure 3: icSHAPE Reveals Structural Profiles Associated with Translationa, Cartoon representation of ribosomes translating an mRNA. The uORF initiation site is represented by ribosome initiation upstream of the canonical start codon. The canonical start position is demarcated by “AUG”. The N-terminal truncation is represented as a ribosome initiating to the 3’-end of the canonical start “AUG”. b, icSHAPE profile at canonical start codon position. c, icSHAPE profiles at uORF and N-terminal truncation sites. d, Cartoon representation of a paused ribosome and its corresponding A-P-E sites. A: acceptor; P: peptidyl-transferase; E: exit. e, The icSHAPE profile at ribosome pause sites. f, icSHAPE profile at negative control sites for pause sequences. Gray box highlights a region of structural difference upstream of true pause sites vs. controls.

Mentions: We hypothesized that translational regulatory elements may have their icSHAPE profiles conserved between in vivo and in vitro because the Kozak sequence, important for translation initiation16, is among the most stable (low VTD) regions within mRNAs (Fig. 2d). RNA accessibility from −1 to −5 nucleotides upstream of the start codon plays a major role in regulating translational output10,17. We used translation initiation18 and pause sites18, defined by ribosome profiling, to center our structural reactivity analysis across the transcriptome (Fig. 3). Canonical initiation AUG sites are indeed preceded by ~5 nts of increased accessibility, and this pattern is nearly identical to in vitro folded RNA (Fig. 3a and 3b). A similar pattern of conserved upstream accessibility also precedes noncanonical start sites at upstream open reading frames (uORF) and N-terminal truncations (Fig. 3c). Non-start site AUG codons are also associated with increased preceding reactivity, while noncanonical CUG start codons have a different profile, suggesting that RNA accessibility alone is not sufficient to dictate translational start sites (Extended Data Fig. 7). Ribosome profiling also defined ribosome pause sites as having a strong preference for glutamate or aspartate in the A-site, where transfer RNA (tRNA) identity and the nascent peptide sequence are believed to strongly influence translation kinetics18. icSHAPE data at ribosome pause sites revealed a distinctive signature: loss of reactivity at the E and P sites while the A site is more reactive, preceded by strong 3-nt periodic reactivity pattern 5’ to the pause site for ~12 nts (Fig. 3d and 3e). Furthermore, a very similar pattern was observed in vitro under conditions that do not maintain mRNA interactions with the ribosome or tRNAs, suggesting that these structural profiles are programmed by mRNA sequence. Analysis of negative control sites – defined as sites on the same transcripts that match the codon composition, are in frame, and are at least 20 nts away from true pause sites – showed a very similar icSHAPE signature at the presumed ribosome E, P, and A sites, but negative controls lacked the 5’ periodic signal (gray box in Fig. 3e and 3f). This observation suggests that the icSHAPE signature at ribosome pause sites is likely due to the codon bias at such sites, but sequences 5’ to the pause may influence pausing. These results identify several physiological structural signatures of translational control elements, and suggest they may be largely pre-programmed by mRNA sequence.


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)

icSHAPE Reveals Structural Profiles Associated with Translationa, Cartoon representation of ribosomes translating an mRNA. The uORF initiation site is represented by ribosome initiation upstream of the canonical start codon. The canonical start position is demarcated by “AUG”. The N-terminal truncation is represented as a ribosome initiating to the 3’-end of the canonical start “AUG”. b, icSHAPE profile at canonical start codon position. c, icSHAPE profiles at uORF and N-terminal truncation sites. d, Cartoon representation of a paused ribosome and its corresponding A-P-E sites. A: acceptor; P: peptidyl-transferase; E: exit. e, The icSHAPE profile at ribosome pause sites. f, icSHAPE profile at negative control sites for pause sequences. Gray box highlights a region of structural difference upstream of true pause sites vs. controls.
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Related In: Results  -  Collection

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Figure 3: icSHAPE Reveals Structural Profiles Associated with Translationa, Cartoon representation of ribosomes translating an mRNA. The uORF initiation site is represented by ribosome initiation upstream of the canonical start codon. The canonical start position is demarcated by “AUG”. The N-terminal truncation is represented as a ribosome initiating to the 3’-end of the canonical start “AUG”. b, icSHAPE profile at canonical start codon position. c, icSHAPE profiles at uORF and N-terminal truncation sites. d, Cartoon representation of a paused ribosome and its corresponding A-P-E sites. A: acceptor; P: peptidyl-transferase; E: exit. e, The icSHAPE profile at ribosome pause sites. f, icSHAPE profile at negative control sites for pause sequences. Gray box highlights a region of structural difference upstream of true pause sites vs. controls.
Mentions: We hypothesized that translational regulatory elements may have their icSHAPE profiles conserved between in vivo and in vitro because the Kozak sequence, important for translation initiation16, is among the most stable (low VTD) regions within mRNAs (Fig. 2d). RNA accessibility from −1 to −5 nucleotides upstream of the start codon plays a major role in regulating translational output10,17. We used translation initiation18 and pause sites18, defined by ribosome profiling, to center our structural reactivity analysis across the transcriptome (Fig. 3). Canonical initiation AUG sites are indeed preceded by ~5 nts of increased accessibility, and this pattern is nearly identical to in vitro folded RNA (Fig. 3a and 3b). A similar pattern of conserved upstream accessibility also precedes noncanonical start sites at upstream open reading frames (uORF) and N-terminal truncations (Fig. 3c). Non-start site AUG codons are also associated with increased preceding reactivity, while noncanonical CUG start codons have a different profile, suggesting that RNA accessibility alone is not sufficient to dictate translational start sites (Extended Data Fig. 7). Ribosome profiling also defined ribosome pause sites as having a strong preference for glutamate or aspartate in the A-site, where transfer RNA (tRNA) identity and the nascent peptide sequence are believed to strongly influence translation kinetics18. icSHAPE data at ribosome pause sites revealed a distinctive signature: loss of reactivity at the E and P sites while the A site is more reactive, preceded by strong 3-nt periodic reactivity pattern 5’ to the pause site for ~12 nts (Fig. 3d and 3e). Furthermore, a very similar pattern was observed in vitro under conditions that do not maintain mRNA interactions with the ribosome or tRNAs, suggesting that these structural profiles are programmed by mRNA sequence. Analysis of negative control sites – defined as sites on the same transcripts that match the codon composition, are in frame, and are at least 20 nts away from true pause sites – showed a very similar icSHAPE signature at the presumed ribosome E, P, and A sites, but negative controls lacked the 5’ periodic signal (gray box in Fig. 3e and 3f). This observation suggests that the icSHAPE signature at ribosome pause sites is likely due to the codon bias at such sites, but sequences 5’ to the pause may influence pausing. These results identify several physiological structural signatures of translational control elements, and suggest they may be largely pre-programmed by mRNA sequence.

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