23 DOWN-REGULATION OF MIR150 PROMOTES THE ACCUMULATION OF CLASSICAL MONOCYTES, A CHARACTERISTIC FEATURE OF CHRONIC MYELOMONOCYTIC LEUKEMIA

Background: Advances in whole exome and RNAsequencing have led to the identification of recurrent mutations in several RNA splicingfactors, such as SRSF2, U2AF1, SF3B1, and ZRSR2. The mechanism by which mutations in splicing factorscontribute to MDS is not yet known. Introduction: Mutations in SRSF2 affect predominantly Proline 95 (P95H/L/R) located within the C-terminal end of the RNAbinding domain. SRSF2 binds to so-called exonic splicing enhancers(ESE) or inhibitors (ESI) within pre-mRNA exons, thus effecting exon inclusion or exclusion. Purpose: We seek to determine how mutations of SRSF2 P95 affect the structure and function of SRSF2 and to discern how mutations of SRSF2 P95 lead to alternative spliceevents that cause myelodysplasia. Materials and Methods: To investigate the role of mutant SRSF2 in the pathogenesis of MDS, we performed isothermal calorimetry (ITC) and nuclear magnetic resonance modeling (NMR) to determine SRSF2 RNAbinding and structure. We performed in vivo RNAimmunoprecipitation to identify bound RNAand we performed RNA deep sequencingto identify alternative spliceevents. Results: Wildtype SRSF2 RRM binds the RNA consensus sequence5’-SSNG-3’ (S=G/C, N=G/C/T/A). It binds 5’-CCNG-3’ and 5’GGNG-3’ equally well with a dissociation constant (Kd) of 0.27 M (Daubner et al. The EMBO Journal (2012) 31, 162–174). SRSF2 P95MUT RRM binds 5’-CCNG-3’ with a Kd=0.06 M, resulting in a ~4-fold increased affinity, while binding to 5’-GGNG-3’ is minimally affected. This leads to an end result of not only altered binding affinity, but also altered RNAbinding specificity. To understand, how mutation of P95 in the C-terminal arm of the RRM, outside the canonical RNAbinding domain, could affect RNAbinding we performed NMR titration experiments. P95 mutations result in significant chemical shift perturbation in the Nand C-termini when bound to 5’-uCCAGu-3’, but not when bound to 5’-uGGAGu-3’, providing a structural basis for altered RNAbinding affinities identified via ITC. To address, whether in vitro RNAbinding affinities translate into altered RNAbinding in vivo we performed High Throughput Sequencing – UV Cross-linking RNAImmunoprecipitation ( HITS-CLIP). HITS-CLIPidentified differentially bound RNA targets. Analysisof sequence enrichment within bound exons confirms preferential binding of 5’-CCNG-3’ consensus sequencesby mutant SRSF2. RNA deep sequencingidentified exon skippingas the most common alternative splicingevent, consistent with SRSF2’s role in alternative splicingvia binding to ESEs. Conclusions: Mutations in SRSF2 identified in myelodysplasia affect RNAbinding affinity, specificity, and alternative splicingoutcomes, likely contributing to the pathogenesis of myelodysplasia.