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Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

Lelyveld VS, Björkbom A, Ransey EM, Sliz P, Szostak JW - J. Am. Chem. Soc. (2015)

Bottom Line: Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking.The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry.Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.

ABSTRACT
High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

No MeSH data available.


Identifying RNA-protein contacts with site-specific RNA mass labeling.(a) Sample preparation workflow. Oligonucleotides are prepared withselectively enriched 18O phosphates at distinct positions(black circle), separately incubated with protein, UV cross-linked,hydrolyzed to peptide-nucleotide fragments, and analyzed by MS. (b) Calculated and observed isotopicdistributionsfor [M – 4H]4– ion of 25-nt preEM-let-7f RNA (GGGGUAGUGAU11UUUACCCUGGAGAU)labeled with 18O at the phosphodiester following U11. (c)Direct LC-MS sequencing of U11-labeled RNA, with the mass labeledposition indicated as U* (red). (d) Isotope distribution of [MGFGFLSMTAR+ UMP + 2H]2+, a tryptic peptide ion derived from cross-linkingLin28A in the presence of preEM-let-7f with natural abundance(native) or 18O mass labeled at the 3′ phosphodiesterfollowing U11 or U12. All spectra are normalized to the abundanceof the monoisotopic ion 771.3 m/z.
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fig1: Identifying RNA-protein contacts with site-specific RNA mass labeling.(a) Sample preparation workflow. Oligonucleotides are prepared withselectively enriched 18O phosphates at distinct positions(black circle), separately incubated with protein, UV cross-linked,hydrolyzed to peptide-nucleotide fragments, and analyzed by MS. (b) Calculated and observed isotopicdistributionsfor [M – 4H]4– ion of 25-nt preEM-let-7f RNA (GGGGUAGUGAU11UUUACCCUGGAGAU)labeled with 18O at the phosphodiester following U11. (c)Direct LC-MS sequencing of U11-labeled RNA, with the mass labeledposition indicated as U* (red). (d) Isotope distribution of [MGFGFLSMTAR+ UMP + 2H]2+, a tryptic peptide ion derived from cross-linkingLin28A in the presence of preEM-let-7f with natural abundance(native) or 18O mass labeled at the 3′ phosphodiesterfollowing U11 or U12. All spectra are normalized to the abundanceof the monoisotopic ion 771.3 m/z.

Mentions: Here we present an alternativeapproach based on site-specificstable isotope labeling that can be used for efficient identificationof interacting sites by liquid chromatography-mass spectrometry (LC-MS).The technique relies on a facile, inexpensive, and fully automatedmethod to generate individual 18O-labeled phosphodiesterlinkages during oligonucleotide synthesis. These site-specificallymass labeled oligonucleotides can be prepared in a straightforwardmanner by standard solid-phase synthesis, such that no cumbersomeorganic synthesis is necessary to generate specific mass labeled DNAor RNA probes. Site-specifically labeled oligonucleotides can be cross-linkedto interacting proteins (Figure 1A), and the resulting covalent product can be subjectedto hydrolysis and analyzed by MS. Covalent nucleotide-labeled peptidesprepared in this manner exhibit a unique isotopic distribution exclusivelywhen an 18O mass label is retained adjacent to the cross-linksite.


Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

Lelyveld VS, Björkbom A, Ransey EM, Sliz P, Szostak JW - J. Am. Chem. Soc. (2015)

Identifying RNA-protein contacts with site-specific RNA mass labeling.(a) Sample preparation workflow. Oligonucleotides are prepared withselectively enriched 18O phosphates at distinct positions(black circle), separately incubated with protein, UV cross-linked,hydrolyzed to peptide-nucleotide fragments, and analyzed by MS. (b) Calculated and observed isotopicdistributionsfor [M – 4H]4– ion of 25-nt preEM-let-7f RNA (GGGGUAGUGAU11UUUACCCUGGAGAU)labeled with 18O at the phosphodiester following U11. (c)Direct LC-MS sequencing of U11-labeled RNA, with the mass labeledposition indicated as U* (red). (d) Isotope distribution of [MGFGFLSMTAR+ UMP + 2H]2+, a tryptic peptide ion derived from cross-linkingLin28A in the presence of preEM-let-7f with natural abundance(native) or 18O mass labeled at the 3′ phosphodiesterfollowing U11 or U12. All spectra are normalized to the abundanceof the monoisotopic ion 771.3 m/z.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697197&req=5

fig1: Identifying RNA-protein contacts with site-specific RNA mass labeling.(a) Sample preparation workflow. Oligonucleotides are prepared withselectively enriched 18O phosphates at distinct positions(black circle), separately incubated with protein, UV cross-linked,hydrolyzed to peptide-nucleotide fragments, and analyzed by MS. (b) Calculated and observed isotopicdistributionsfor [M – 4H]4– ion of 25-nt preEM-let-7f RNA (GGGGUAGUGAU11UUUACCCUGGAGAU)labeled with 18O at the phosphodiester following U11. (c)Direct LC-MS sequencing of U11-labeled RNA, with the mass labeledposition indicated as U* (red). (d) Isotope distribution of [MGFGFLSMTAR+ UMP + 2H]2+, a tryptic peptide ion derived from cross-linkingLin28A in the presence of preEM-let-7f with natural abundance(native) or 18O mass labeled at the 3′ phosphodiesterfollowing U11 or U12. All spectra are normalized to the abundanceof the monoisotopic ion 771.3 m/z.
Mentions: Here we present an alternativeapproach based on site-specificstable isotope labeling that can be used for efficient identificationof interacting sites by liquid chromatography-mass spectrometry (LC-MS).The technique relies on a facile, inexpensive, and fully automatedmethod to generate individual 18O-labeled phosphodiesterlinkages during oligonucleotide synthesis. These site-specificallymass labeled oligonucleotides can be prepared in a straightforwardmanner by standard solid-phase synthesis, such that no cumbersomeorganic synthesis is necessary to generate specific mass labeled DNAor RNA probes. Site-specifically labeled oligonucleotides can be cross-linkedto interacting proteins (Figure 1A), and the resulting covalent product can be subjectedto hydrolysis and analyzed by MS. Covalent nucleotide-labeled peptidesprepared in this manner exhibit a unique isotopic distribution exclusivelywhen an 18O mass label is retained adjacent to the cross-linksite.

Bottom Line: Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking.The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry.Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.

ABSTRACT
High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

No MeSH data available.