Departments of Developmental and Molecular Biology and Medicine (Oncology), Gottesman Institute of Stem Cell Biology and Regenerative Medicine
Splicing and transcription are tightly intertwined processes that occur simultaneously. Disruption of the spliceosome or inhibition of splicing in cultured cells and yeast leads to transcriptional stalling, suggesting the existence of a transcriptional checkpoint that is linked to splicing. It is unclear if this occurs in vivo and if there are specific phenotypic consequences associated with activating a splicing checkpoint. To investigate if disruption of the spliceosome affects transcriptional elongation in vivo, we utilized a zebrafish loss-of-function mutant for the core spliceosomal component sf3b1 (splicing factor, 3b subunit 1). In order to detect transcriptional stalling, we conducted Pol II ChIP-seq of wild type and mutant embryos, and are analyzing the results to determine if the position of RNA PolII across gene bodies is altered. To corroborate the Pol II ChIP-seq results, we will also analyze nascent RNA lengths in genes that are misexpressed in sf3b1 mutants. Additionally, sf3b1 mutants have defects in hematopoietic stem and progenitor cell (HSPC) formation from hemogenic endothelial cells. HSPCs require high transcriptional levels to arise from endothelium.To determine if there is transcriptional impairment in the hemogenic endothelium in the mutant, we employed flow cytometry to measure the amount of 5-ethynyluridine (EU) incorporation into RNA in flk1:gfp positive endothelial cells in wild-type and sf3b1 siblings. The mutant endothelial cells had two-fold less EU incorporation than their wild-type siblings indicating that loss of sf3b1 leads to a diminished transcriptional output. These studies will help determine if spliceosomal disruption causes transcriptional stalling in vivo, and whether the HSPC formation defects observed in sf3b1 mutants are caused by transcriptional deficiencies.