Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-04-2019Alternative splicing is a fundamental mechanism that allows the generation of
multiple isoforms of the same pre-mRNA. Although splicing is a necessary process in all
eukaryotes from yeast to mammals, alternative splicing is more prevalent in higher than
lower eukaryotes, and especially in vertebrates. Nearly 95% of mammalian genes undergo
alternative splicing, so mis-regulation of this process may contribute to the development of
different diseases such as cancer. This higher level of regulation requires additional proteins
to facilitate the correct positioning of the spliceosomal subunits on the primary transcript.
Here, we reveal a role for DIDO3, one of the three Dido gene products, in SFPQ binding and
alternative splicing. Previous studies in our laboratory established binding to H3K4Me3
through a PHD domain located at the amino terminal region of the protein. A TFS2M domain
at the central part of DIDO3 promotes association with the RNAPII jaw during transcription
elongation. Now, we show interaction between the carboxy-terminus of DIDO3 and SFPQ, a
known splicing factor. SFPQ is a protein that associates with the polypyrimidine tract, and in
particular facilitates correct U2 snRNP positioning on the 3’ splice site of exons. The
generation of a Dido mutant lacking DIDO3 while preserving the other two isoforms
suppressed binding of SFPQ to RNA and increased skipping for a large general group of
exons. Exons containing SRSF1 recognition sequences however were included more
efficiently. Alternative splicing was also studied in the context of another Dido mutant. The
deletion of the amino terminal region of DIDO3, too, resulted in alternatively spliced exons,
although the observed splicing defects were milder. In this mutant, downstream T-rich
regions, associated with RNA polymerase II pause sites, facilitated the inclusion of their
upstream exons.
Together, our results indicate that the DIDO3-SFPQ association regulates alternative
splicing. Due to its modular structure, DIDO3 could act as a bridge between RNA polymerase
II and SFPQ, and thereby control the recruitment of the latter to the nascent RNA. Lack of
the DIDO3-specific domain reduces SFPQ availability for the RNA and promotes the skipping
of exons that are highly SFPQ-dependent. Although alternative pathways also regulate exon
inclusion or skipping, we propose that the DIDO3-SFPQ axis in particular has evolved to
expand alternative splicing regulation and maintain RNA splicing efficiency in mammals