Identification of SRSF3 binding sites in megakaryocytes

Abstract

RNA-binding proteins (RBPs) play essential roles in post-transcriptional gene regulation, influencing mRNA splicing, localization, stability, and translation. In megakaryocytes, these processes are critical for proper differentiation and platelet biogenesis. This thesis focused on optimizing a modified version of the crosslinking and immunoprecipitation (CLIP) method, known as streamlined individual nucleotide resolution CLIP (siCLIP), to study RNA–protein interactions in megakaryocytes, with a particular emphasis on Serine-arginine rich splicing factor 3 (SRSF3).To achieve this, a MEG-01 megakaryocytic cell line was genome-edited using CRISPR-Cas9 to integrate a HaloTag at the endogenous SRSF3 locus, which enables the isolation of SRSF3–RNA complexes. Integration was confirmed by PCR genotyping and confocal microscopy. Several critical parameters of siCLIP were optimized, including starting cell number, RNase I digestion conditions, and PCR amplification cycles, to ensure the recovery of high-quality and proper RNA fragments. The results showed that predominantly short RNA fragments were recovered that may have resulted from technical factors or reactions conditions. While the optimized protocol demonstrated offered insights into the preparation steps of siCLIP, further refinement is needed before transcriptome-wide mapping of SRSF3 binding sites can be pursued through high-throughput sequencing. The siCLIP could provide a robust methodological platform for investigating RBP–RNA interactions in hematopoietic cells and contributes to the broader understanding of post-transcriptional regulation in megakaryocyte biogenesis and platelet formation.