Protein ubiquitination, one of the most common posttranslational modifications, is involved in numerous cellular processes. It shows remarkable topological and functional diversity, a phenomenon known as the ubiquitin code, through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understanding the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of

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... itin in both human and yeast cells (Tsuchiya et al., 2018). Despite previous studies, the lack of specific binders as tools to detect K29-linked polyubiquitin has prevented further understanding of its function. In this study, we screened and characterized a synthetic antigen-binding fragment (named sAB-K29) that can specifically recognize K29-linked polyubiquitin. We determined the crystal structure of sAB-K29 bound to K29-linked diubiquitin (diUb), which revealed the molecular basis of sAB-K29 specificity. Using sAB-K29 as a tool, we identified the involvement of K29-linked ubiquitination in RNA processing, the proteotoxic stress response and cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody at the telophase of mitosis and that downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.