The role of FUBP1 in splicing, disease and evolution
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Abstract
Splicing is the process of intron excision and exon re-ligation, and it is a crucial step of gene
regulation. Mechanistically, splicing is well studied. However, splicing is a highly regulated process
and there are multitudes of cis-regulatory and trans-acting elements that control splicing. This socalled
“splicing code” is less understood, and in this study, we employed an array of high-throughput
sequencing methods, coupled to structural biology, mathematical modeling and molecular biological
experiments, to examine the role of the Far-Upstream Binding Protein FUBP1 in splicing and further,
the regulation of CD19 mRNA splicing.
Previous studies showed that the transcription factor FUBP1 also regulates certain splicing events.
Additionally, demonstrated that FUBP1 stabilizes U2AF2, a core splicing component, to
the py-tract. So far, however, the transcriptome-wide role of FUBP1 in splicing remains elusive. We
showed that FUBP1 binds to a hitherto unknown U-rich sequence upstream of the branch point. It
binds more than 80% of all pre-mRNA, rendering it a core splicing factor. Using NMR-based
technologies and reporter assays, we showed that FUBP1 interacts with proteins of both the 3’ss and
the 5’ss. We found that the N-terminal N-box of FUBP1 interacts with the RRM2 domain of U2AF2.
In addition, an animal-specific C-terminal A/B-box of FUBP1 interacts with proline-rich stretches
of SF1. RNA-seq of two FUBP1 mutant cell lines generated by CRISPR/Cas9 revealed that exons
flanked by long introns are skipped when FUBP1 is lost. Analyzing RNA-seq of low-grade glioma
patients with FUBP1 deficiencies yielded comparable results, indicating that FUBP1 facilitates
splicing of long introns. We hypothesize that FUBP1 evolved to regulate splicing of long introns in
higher eukaryotes by interacting with both the 3’ss and the 5’ss, connecting the splice sites.
In a second study, we dissected the elements that are important for splicing of CD19 mRNA. B cell
acute lymphoblastic leukemia (B-ALL) patients can be treated with the cell-based immunotherapy
CART-19, which specifically targets CD19-expressing cells. However, they often experience relapse
due to CD19 epitope loss. Using a high-throughput mutagenesis approach, we detected over 200
mutations that lead to approx. 100 cryptic isoforms that can contribute to epitope loss. By cloning a
selection of the mutations into a CD19 minigene and performing RT-PCR, we could confirm these
results. In addition, shRNA-induced KD of splicing factors revealed that trans-acting factors like
SF3B4 and PTBP1 are essential for correct CD19 splicing. With this study, we offer a comprehensive
overview of the CD19 splicing code.
Taken together, this PhD thesis highlights the interdependency between cis- and trans-acting
splicing components. It underlines the importance of splicing – and the knowledge thereof – in the
context of health and disease.