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

Abstract

Background: Advances in whole exome and RNA sequencing have led to the identification of recurrent mutations in several RNA splicing factors, such as SRSF2, U2AF1, SF3B1, and ZRSR2. The mechanism by which mutations in splicing factors contribute 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 RNA binding 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 splice events 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 RNA binding and structure. We performed in vivo RNA immunoprecipitation to identify bound RNA and we performed RNA deep sequencing to identify alternative splice events. Results: Wildtype SRSF2 RRM binds the RNA consensus sequence 5’-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 RNA binding specificity. To understand, how mutation of P95 in the C-terminal arm of the RRM, outside the canonical RNA binding domain, could affect RNA binding 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 RNA binding affinities identified via ITC. To address, whether in vitro RNA binding affinities translate into altered RNA binding in vivo we performed High Throughput Sequencing – UV Cross-linking RNA Immunoprecipitation (HITS-CLIP). HITS-CLIP identified differentially bound RNA targets. Analysis of sequence enrichment within bound exons confirms preferential binding of 5’-CCNG-3’ consensus sequences by mutant SRSF2. RNA deep sequencing identified exon skipping as the most common alternative splicing event, consistent with SRSF2’s role in alternative splicing via binding to ESEs. Conclusions: Mutations in SRSF2 identified in myelodysplasia affect RNA binding affinity, specificity, and alternative splicing outcomes, likely contributing to the pathogenesis of myelodysplasia.