Journal
EMBO REPORTS
Volume 12, Issue 7, Pages 690-696Publisher
WILEY
DOI: 10.1038/embor.2011.100
Keywords
single-molecule fluorescence; single-molecule immunoprecipitation; TUT4 (ZCCHC11); Lin28 (Lin28a, Lin28b, Lin28); real-time hybridization
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Funding
- National Research Foundation of Korea [20090063603, 20090081562]
- Ministry of Education, Science and Technology of Korea
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Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.
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