Functional dissection of SPOP on the amino acid level reveals a comprehensive functional landscape of variants during tumorigenesis

Numerous proteins display pleiotropic functions in different clinical contexts. However, molecular mechanism underlying such effects is rarely understood. Speckle-type POZ protein (SPOP) is a typical example, exhibiting tumor-suppressing or -promoting effects in different tumor types in accordance with different amino acid changes; specifically, two distinct sets of variants in SPOP are commonly found in subsets of prostate cancer and endometrial cancer patients. To comprehensively characterize the functional landscape of SPOP alteration, we performed a deep mutational screening (DMS), elucidating the functionality of 7,933 out of 8,228 possible single amino acid changes (96.4% coverage). Leveraging the observation that overexpression of human SPOP leads to yeast growth arrest, we assessed the functionality of each variant using a yeast proliferation assay. In addition, our approach combined long-read and short-read sequencing. While long-read-based scores demonstrated improved concordance with functional classification, both platforms demonstrated a largely consistent classification pattern of external data into the same tiers. Finally, our DMS model enables a clear distinction between loss-of-function and putative gain-of-function changes and reveals their differential characteristics in both protein structure and genetic assessments. These results demonstrate the utility of our approach in high resolution mapping and amino-acid-level interpretation of protein function.