Alternative splicing of pre-messenger RNA increases genetic diversity and recent studies estimate that most human multi-exon genes are alternatively spliced. If this process is not highly regulated and accurate, it leads to mis-splicing events which may result in proteins with altered function. A growing body of work has implicated mis-splicing events as contributing to a wide range of diseases including cancer, neurodegenerative diseases, and muscular dystrophies. Understanding the mechanisms that cause aberrant splicing events and how this leads to disease is vital for designing effective therapeutic strategies. This review focuses on advances in therapies targeting splicing and highlights the animal models developed to recapitulate disease phenotypes as a model for testing these therapies.

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA analogue element called the HomolD box. The HomolD‐binding protein Rrn7 forms a complex with the RNA polymerase II machinery. Despite the importance of ribosome biogenesis to cell survival, the mechanisms involved in the regulation of transcription of eukaryotic RPGs are unknown. In this study, we identified Rrn7 as a new substrate of the pleiotropic casein kinase 2 (CK2), which is a regulator of basal transcription. Recombinant Rrn7 from S. pombe, which is often used as a model organism for studying eukaryotic transcription, interacted with CK2 in vitro and in vivo. Furthermore, CK2‐mediated phosphorylation of Rrn7 inhibited its HomolD‐directed transcriptional activity and ability to bind to an oligonucleotide containing a HomolD box in vitro. Mutation of Rrn7 at Thr67 abolished these effects, indicating that this residue is a critical CK2 phosphorylation site. Finally, Rrn7 interacted with the regulatory subunit of CK2 in vivo, inhibition of CK2 in vivo potentiated ribosomal protein gene transcription, and chromatin immunoprecipitation analyses identified that the catalytic subunit of CK2 was associated with the rpk5 gene promoter in S. pombe. Taken together, these data suggest that CK2 inhibits ribosomal protein gene transcription in S. pombe via phosphorylation of Rrn7 at Thr67.

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.

◥MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knock-down induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2.Implications: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress. Materials and MethodsCell culture, growth, and transfection conditions MCF7, NIH 3T3 cells, HeLa, and C2C12 cells were obtained from ATCC and Rh18/SMS-CTR cells were obtained from Peter J. Houghton (Greehey Children's Cancer Research Institute, San Antonio, TX). HeLa S3 cells were obtained from Hua Lou (Case Western University, Cleveland, OH). MCF7 and HeLa cells were chosen because these cells lines have been studied extensively from the standpoint of MDM2 alternative splicing and because they are robust in their tolerance to genotoxic stress, as well as amenable to genetic manipulation by transfection. SMS-CTR and Rh18 cells were