title: CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.
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.
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Tyler
authors.initials: T
authors.lastname: Funnell
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Tasaki
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.|Janssen Pharmaceuticals, Toronto, Ontario, Canada.
authors.forename: Arusha
authors.initials: A
authors.lastname: Oloumi
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Araki
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Esther
authors.initials: E
authors.lastname: Kong
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Damian
authors.identifier: 0000-0002-5370-4592
authors.initials: D
authors.lastname: Yap
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakayama
authors.affiliation: Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.
authors.forename: Christopher S
authors.identifier: 0000-0002-6980-4459
authors.initials: CS
authors.lastname: Hughes
authors.affiliation: Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.
authors.forename: S-W Grace
authors.initials: SG
authors.lastname: Cheng
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tozaki
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Misa
authors.initials: M
authors.lastname: Iwatani
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Miyazaki
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Nao
authors.initials: N
authors.lastname: Morishita
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Morishita
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Ogasawara-Shimizu
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Momoko
authors.initials: M
authors.lastname: Ohori
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Nakao
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Karashima
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Sano
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Murai
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Uchiyama
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kawamoto
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.|Fujirebio Inc., Tokyo, Japan.
authors.forename: Ryujiro
authors.initials: R
authors.lastname: Hara
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.|Department of Innovative Drug Discovery and Development, Japan Agency for Medical Research and Development, Osaka, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nakanishi
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Karey
authors.initials: K
authors.lastname: Shumansky
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.|Advanced Research Computing, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jamie
authors.initials: J
authors.lastname: Rosner
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Adrian
authors.initials: A
authors.lastname: Wan
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Steven
authors.initials: S
authors.lastname: McKinney
authors.affiliation: Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6H 3N1.
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Morin
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakanishi
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5.
authors.forename: Sohrab
authors.initials: S
authors.lastname: Shah
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan. hiroyoshi.toyoshiba@takeda.com.
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Toyoshiba
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3. saparicio@bccrc.ca.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 2B5. saparicio@bccrc.ca.
authors.forename: Samuel
authors.initials: S
authors.lastname: Aparicio
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D005091:Exons; D020869:Gene Expression Profiling; D015894:Genome, Human; D045325:HCT116 Cells; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000138:chemical synthesis / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
chemical_list: D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases