CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor

  • Nat Commun. 2017 Feb 23;8(1):7. doi: 10.1038/s41467-016-0008-7.
Tyler Funnell  1  2 ,  Shinya Tasaki  3 ,  Arusha Oloumi  1  2  4 ,  Shinsuke Araki  3 ,  Esther Kong  1  2 ,  Damian Yap  1  2 ,  Yusuke Nakayama  3 ,  Christopher S Hughes  5 ,  S-W Grace Cheng  5 ,  Hirokazu Tozaki  3 ,  Misa Iwatani  3 ,  Satoshi Sasaki  3 ,  Tomohiro Ohashi  3 ,  Tohru Miyazaki  3 ,  Nao Morishita  3 ,  Daisuke Morishita  3 ,  Mari Ogasawara-Shimizu  3 ,  Momoko Ohori  3 ,  Shoichi Nakao  3 ,  Masatoshi Karashima  3 ,  Masaya Sano  3 ,  Aiko Murai  3 ,  Toshiyuki Nomura  3 ,  Noriko Uchiyama  3 ,  Tomohiro Kawamoto  3 ,  Ryujiro Hara  3  6 ,  Osamu Nakanishi  3  7 ,  Karey Shumansky  1  2 ,  Jamie Rosner  1  2  8 ,  Adrian Wan  1  2 ,  Steven McKinney  1  2 ,  Gregg B Morin  5  9 ,  Atsushi Nakanishi  3 ,  Sohrab Shah  1  2 ,  Hiroyoshi Toyoshiba  10 ,  Samuel Aparicio  11  12
Affiliations
  • 1. Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.
  • 2. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
  • 3. Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
  • 4. Janssen Pharmaceuticals, Toronto, Ontario, Canada.
  • 5. Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.
  • 6. Fujirebio Inc., Tokyo, Japan.
  • 7. Department of Innovative Drug Discovery and Development, Japan Agency for Medical Research and Development, Osaka, Japan.
  • 8. Advanced Research Computing, University of British Columbia, Vancouver, British Columbia, Canada.
  • 9. Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6H 3N1.
  • 10. Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan. [email protected].
  • 11. Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3. [email protected].
  • 12. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5. [email protected].
PMID: 28232751 DOI: 10.1038/s41467-016-0008-7
Abstract

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK Inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

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