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

Numerous proteins display pleiotropic functions in different clinical contexts. However, molecular mechanism underlying such effects is rarely understood. The Speckle-type POZ protein (SPOP) is a typical example, showing tumor-suppressing or promoting effects due to different amino acid changes in different tumor types. Specifically, two distinct sets of variants in SPOP are commonly found in subsets of prostate cancer or endometrial cancer patients. To comprehensively characterize the functional landscape of SPOP alteration, we performed a deep mutational screening, elucidating functionality of 7,933 of 8,228 possible single amino acid changes (96.4% coverage). Utilizing 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 assessing these variants, we combined long-read and short-read sequencing reads. While long-read-based scores demonstrated improved concordance with functional classification, both platforms demonstrated a largely consistent classification pattern of external dataset with prediction score. Our DMS model enables a clear distinction between loss-of-function and putative gain-of-function, and reveals their differential characteristics in both protein structure and our functional validation. Our approach demonstrates the utility of our approach in high resolution mapping and amino acid-level interpretation of protein function.