Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells
Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2022.1062448/full |
| _version_ | 1817971304509210624 |
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| author | Roberto Giambruno Roberto Giambruno Francesco Nicassio |
| author_facet | Roberto Giambruno Roberto Giambruno Francesco Nicassio |
| author_sort | Roberto Giambruno |
| collection | DOAJ |
| description | Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions. |
| first_indexed | 2024-04-13T20:45:05Z |
| format | Article |
| id | doaj.art-172bf44cf4a04271b137aad11d76f56b |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-13T20:45:05Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-172bf44cf4a04271b137aad11d76f56b2022-12-22T02:30:43ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2022-11-01910.3389/fmolb.2022.10624481062448Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cellsRoberto Giambruno0Roberto Giambruno1Francesco Nicassio2Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Milano, ItalyInstitute of Biomedical Technologies, National Research Council, Segrate, ItalyCenter for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Milano, ItalyProximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions.https://www.frontiersin.org/articles/10.3389/fmolb.2022.1062448/fullprotein-RNA interactionsproximity biotinylationAPEX2affinity purificationaffinity purification coupled to mass spectrometryAPEX-seq |
| spellingShingle | Roberto Giambruno Roberto Giambruno Francesco Nicassio Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells Frontiers in Molecular Biosciences protein-RNA interactions proximity biotinylation APEX2 affinity purification affinity purification coupled to mass spectrometry APEX-seq |
| title | Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells |
| title_full | Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells |
| title_fullStr | Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells |
| title_full_unstemmed | Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells |
| title_short | Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells |
| title_sort | proximity dependent biotinylation technologies for mapping rna protein interactions in live cells |
| topic | protein-RNA interactions proximity biotinylation APEX2 affinity purification affinity purification coupled to mass spectrometry APEX-seq |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2022.1062448/full |
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