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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.


Related in: MedlinePlus

Simultaneous assignment of cross-linked nucleotide and amino acidsequence position by MS/MS. (a) A subset of product ions (840–1420 m/z) generated by CID at 20 V from theselected ion 771.3130 m/z (z = 2, isolation width 4 m/z), showing a fragmentation pattern consistent with uridine monophosphate(Up) attached by a mass-neutral linkage to phenylalanine in the fifthsequence position of the peptide MGFGFLSMTAR from Lin28A. Green y-ions are those derived from peptide fragmentation followingloss of Up, shown fully in panel (h). Inset: Full range of productions showing region (dotted box) magnified in the main panel. (b–g)Selected product ions arising from samples prepared with 18O labeling after position U11 (red) or U12 (blue) in preEM-let-7f, with apparent +2 Da enrichment derived only from U11 products.(b) Isotope distribution of remaining unfragmented selected ion (blackdiamond) magnified from the spectra in (a). (c–g) Magnifiedviews of the product ion isotope distributions in panel (a). (h) Fullscan of product ions from CID at 30 V with the same selected ion (blackdiamond), showing peptide sequence fragments for y1–11 andb2–4 arising from Up loss, with [Up – PO4H2]1+ shown as a prominent product ion. Intensitiesin panels (b–g) are normalized to the first isotope.
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fig2: Simultaneous assignment of cross-linked nucleotide and amino acidsequence position by MS/MS. (a) A subset of product ions (840–1420 m/z) generated by CID at 20 V from theselected ion 771.3130 m/z (z = 2, isolation width 4 m/z), showing a fragmentation pattern consistent with uridine monophosphate(Up) attached by a mass-neutral linkage to phenylalanine in the fifthsequence position of the peptide MGFGFLSMTAR from Lin28A. Green y-ions are those derived from peptide fragmentation followingloss of Up, shown fully in panel (h). Inset: Full range of productions showing region (dotted box) magnified in the main panel. (b–g)Selected product ions arising from samples prepared with 18O labeling after position U11 (red) or U12 (blue) in preEM-let-7f, with apparent +2 Da enrichment derived only from U11 products.(b) Isotope distribution of remaining unfragmented selected ion (blackdiamond) magnified from the spectra in (a). (c–g) Magnifiedviews of the product ion isotope distributions in panel (a). (h) Fullscan of product ions from CID at 30 V with the same selected ion (blackdiamond), showing peptide sequence fragments for y1–11 andb2–4 arising from Up loss, with [Up – PO4H2]1+ shown as a prominent product ion. Intensitiesin panels (b–g) are normalized to the first isotope.

Mentions: While the labeled nonbridging phosphodiester oxygens are stableunder biological conditions,15 we notedthat phosphodiester hydrolysis of mass labeled RNA can result in labelexchange with bulk solvent17 when the newlygenerated nucleotide carries a 3′(2′)-monophosphate,which would result in label loss. Acid hydrolysis of RNA generatesa terminal 3′(2′)-monophosphate product,18 and we therefore sought to minimize the impactof exchange when generating digests by this method. We compared 50%(v/v) FA:water (∼12 N) digestion at 60 and 80 °C overtime (Figure S1). The ratio of 16O:18O UMP increased steadily over digest time at 80 °C,indicating significant phosphate oxygen exchange, such that the +2Da species was nearly at natural abundance levels after 2 h. Underthese conditions, the first-order rate constant for exchange was 1.1h–1. When digested at 60 °C, the 18O mass label was well retained even after 2.5 h of digestion, witha significantly slower exchange rate of 0.28 h–1. Alternatively, enzymatic RNA digestion with nuclease P1, whichleaves a 5′-phosphorylated product after cleavage, resultedin negligible loss of the mass label (Figure S3). For the cross-linked RNA-peptide species analyzed in Figures 1d, 2, and S2, we chose a 2 h RNA digestin 50% (v/v) FA at 60 °C.


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)

Simultaneous assignment of cross-linked nucleotide and amino acidsequence position by MS/MS. (a) A subset of product ions (840–1420 m/z) generated by CID at 20 V from theselected ion 771.3130 m/z (z = 2, isolation width 4 m/z), showing a fragmentation pattern consistent with uridine monophosphate(Up) attached by a mass-neutral linkage to phenylalanine in the fifthsequence position of the peptide MGFGFLSMTAR from Lin28A. Green y-ions are those derived from peptide fragmentation followingloss of Up, shown fully in panel (h). Inset: Full range of productions showing region (dotted box) magnified in the main panel. (b–g)Selected product ions arising from samples prepared with 18O labeling after position U11 (red) or U12 (blue) in preEM-let-7f, with apparent +2 Da enrichment derived only from U11 products.(b) Isotope distribution of remaining unfragmented selected ion (blackdiamond) magnified from the spectra in (a). (c–g) Magnifiedviews of the product ion isotope distributions in panel (a). (h) Fullscan of product ions from CID at 30 V with the same selected ion (blackdiamond), showing peptide sequence fragments for y1–11 andb2–4 arising from Up loss, with [Up – PO4H2]1+ shown as a prominent product ion. Intensitiesin panels (b–g) are normalized to the first isotope.
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Related In: Results  -  Collection

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fig2: Simultaneous assignment of cross-linked nucleotide and amino acidsequence position by MS/MS. (a) A subset of product ions (840–1420 m/z) generated by CID at 20 V from theselected ion 771.3130 m/z (z = 2, isolation width 4 m/z), showing a fragmentation pattern consistent with uridine monophosphate(Up) attached by a mass-neutral linkage to phenylalanine in the fifthsequence position of the peptide MGFGFLSMTAR from Lin28A. Green y-ions are those derived from peptide fragmentation followingloss of Up, shown fully in panel (h). Inset: Full range of productions showing region (dotted box) magnified in the main panel. (b–g)Selected product ions arising from samples prepared with 18O labeling after position U11 (red) or U12 (blue) in preEM-let-7f, with apparent +2 Da enrichment derived only from U11 products.(b) Isotope distribution of remaining unfragmented selected ion (blackdiamond) magnified from the spectra in (a). (c–g) Magnifiedviews of the product ion isotope distributions in panel (a). (h) Fullscan of product ions from CID at 30 V with the same selected ion (blackdiamond), showing peptide sequence fragments for y1–11 andb2–4 arising from Up loss, with [Up – PO4H2]1+ shown as a prominent product ion. Intensitiesin panels (b–g) are normalized to the first isotope.
Mentions: While the labeled nonbridging phosphodiester oxygens are stableunder biological conditions,15 we notedthat phosphodiester hydrolysis of mass labeled RNA can result in labelexchange with bulk solvent17 when the newlygenerated nucleotide carries a 3′(2′)-monophosphate,which would result in label loss. Acid hydrolysis of RNA generatesa terminal 3′(2′)-monophosphate product,18 and we therefore sought to minimize the impactof exchange when generating digests by this method. We compared 50%(v/v) FA:water (∼12 N) digestion at 60 and 80 °C overtime (Figure S1). The ratio of 16O:18O UMP increased steadily over digest time at 80 °C,indicating significant phosphate oxygen exchange, such that the +2Da species was nearly at natural abundance levels after 2 h. Underthese conditions, the first-order rate constant for exchange was 1.1h–1. When digested at 60 °C, the 18O mass label was well retained even after 2.5 h of digestion, witha significantly slower exchange rate of 0.28 h–1. Alternatively, enzymatic RNA digestion with nuclease P1, whichleaves a 5′-phosphorylated product after cleavage, resultedin negligible loss of the mass label (Figure S3). For the cross-linked RNA-peptide species analyzed in Figures 1d, 2, and S2, we chose a 2 h RNA digestin 50% (v/v) FA at 60 °C.

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.


Related in: MedlinePlus