Abstract
Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA–protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein–protein crosslinkings. This enabled us, for the first time, to “capture” Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein–protein contacts within an RNA–protein complex.
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Acknowledgments
We thank C.-M. Lin and L.-C. Chang for assistances during the earlier phase of the project; R. Reed for providing pAdML-M3; J. Vilardell for pMS2-MBP; H.-T. Chen for pLH157 and BY4705 strain; S.-C. Cheng for anti-Prp8p and BJ2168 strain; and H.-T. Chen and S.-C. Cheng for insightful discussions. This project has been supported by grants from Ministry of Science and Technology (101-2311-B-001-005 and 102-2311-B-001-029), Thematic Projects (Academia Sinica; AS-99-TP-B20 and AS-103-TP-B12), and Academia Sinica.
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Yeh, FL., Tung, L., Chang, TH. (2016). Detection of Protein–Protein Interaction Within an RNA–Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 1421. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3591-8_15
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DOI: https://doi.org/10.1007/978-1-4939-3591-8_15
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