Myeloid malignancies including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34⁺ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2‐M phase arrest and induction of apoptosis. U2AF1 mutations, on the other hand, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo‐monocytic differentiation towards monocytes but elicit diverse effects on megakaryo‐erythroid differentiation. The SRSF2 mutations skew differentiation towards megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context‐specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. © AlphaMed Press 2018

During splicing of pre-mRNA, 5′ and 3′ splice sites are brought within proximity by interactions between the pre-mRNA bound U1 and U2 snRNPs, followed by recruitment of the tri-snRNP for assembly of the mature spliceosome. Previously, we identified an interaction between the U2 snRNP-specific protein SF3A1 and the stem–loop 4 (SL4) of the U1 snRNA that occurs during the early steps of spliceosome assembly. Although harboring many annotated domains, SF3A1 lacks a canonical RNA binding domain. To identify the U1-SL4 binding region in SF3A1, we expressed amino- and carboxy-terminal deletion constructs using a HeLa cell-based cell free expression system. UV-crosslinking of the truncated proteins with ³²P-U1-SL4 RNA identified the carboxy-terminal ubiquitin-like (UBL) domain of SF3A1 as the RNA binding region. Characterization of the interaction between SF3A1-UBL and U1-SL4 by electrophoretic mobility shift assay and surface plasmon resonance determined high binding affinity (K_D = ∼97 nM), and revealed the double-stranded G-C rich stem of U1-SL4 as an important feature for binding to the UBL domain. Further, mutations of two conserved tyrosine residues, Y772 and Y773, were found to cause a two- and fivefold decrease in the binding affinity for U1-SL4, respectively. Finally, we found that SF3A1-UBL can specifically pull down the U1 snRNP from HeLa nuclear extract, demonstrating its capacity to bind U1-SL4 in the context of the intact snRNP. Thus, the data show that the UBL domain of SF3A1 can function as an RNA binding domain and that mutations in this region may interfere with U1-SL4 binding.

Studying protein interactions in high throughput (HTP) is critical for advancing many aspects of drug discovery, biomarker identification, and diagnostic development. However, existing methods for producing functional protein libraries are costly, time-consuming, and lack real-time kinetic screening capabilities. To address these limitations, we developed an automated platform for HTP production and screening of a library of proteins on biosensor surfaces, facilitating large-scale measurement of interaction kinetics. This technology, Sensor-Integrated Proteome On Chip (SPOC®), involves cell-free protein expression in nanowells directly from customizable libraries of DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins or proteoforms onto a single gold biosensor chip. SPOC protein biosensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of SPOC proteins was validated using a variety of assays. Monoclonal antibodies were found to selectively bind their SPOC protein targets and anti-RBD antibody was used to demonstrate discriminated binding kinetics to numerous SARS-CoV-2 RBD variants on-chip. With advantages of HTP, flexibility, low-cost, and real-time kinetic profiling, the SPOC platform addresses challenges of interrogating protein interactions at scale for research and clinical applications.

Accurate detection of biomolecular interactions is essential in many areas, from the detection of the presence of biomarkers in the clinic to the development of therapeutic drugs and biologics in biopharma to the understanding of various biological processes in basic research. Traditional endpoint approaches can suffer from false-negative results for biomolecular interactions with fast kinetics. By contrast, real-time detection techniques like surface plasmon resonance (SPR) monitor interactions as they form and disassemble, reducing the risk of false-negative results. By leveraging cell-free expressed proteins captured on either glass or SPR biosensors and using two different commercial antibodies with variable off-rates that both target HaloTag antigens as a model, we compare and contrast results from a fluorescence endpoint assay versus real-time sensor-integrated proteome on chip (SPOC^®) SPR-based detection. In this study, we illustrate the limitations of the representative immunofluorescent endpoint assay when investigating transient interactions characterized by fast dissociation rates. We highlight the importance of choosing reagents well suited to the selected assay, as well as the importance of considering binding kinetics and protein ligand conformational states when interpreting results from binding assays, especially for applications as critical as the off-target screening of therapeutics.

An automated proteomic platform for producing and screening an array of functional proteins on biosensor surfaces was developed to address the challenges of measuring proteomic interaction kinetics in high throughput (HTP). This technology is termed Sensor-Integrated Proteome On Chip (SPOC®) which involves in-situ cell-free protein expression in nano-liter volume wells (nanowells) directly from rapidly customizable arrays of plasmid DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins onto a 1.5 sq-cm surface of a single gold biosensor chip. Arrayed SPOC sensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic affinity, avidity data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of the SPOC protein array was validated by antibody binding assay, post-translational modification, mutation-mediated differential binding kinetics, and catalytic activity screening on model SPOC protein arrays containing p53, Src, Jun, Fos, HIST1H3A, and SARS-CoV-2 receptor binding domain (RBD) protein variants of interest, among others. Monoclonal antibodies were found to selectively bind their target proteins on the SPOC array. A commercial anti-RBD antibody was used to demonstrate discriminatory binding to numerous SARS-CoV-2 RBD variants of concern with comprehensive kinetic information. With advantages of HTP, flexibility, low-cost, quick turnaround time, and real-time kinetic affinity profiling, the SPOC proteomic platform addresses the challenges of interrogating protein interactions at scale and can be deployed in various research and clinical applications.