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.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / drug effects*
  • Exons
  • Gene Expression Profiling
  • Genome, Human
  • HCT116 Cells
  • Humans
  • Imidazoles / chemical synthesis
  • Imidazoles / pharmacology*
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / chemical synthesis
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / genetics*
  • Protein Serine-Threonine Kinases / metabolism
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / genetics*
  • Protein-Tyrosine Kinases / metabolism
  • Pyrimidines / chemical synthesis
  • Pyrimidines / pharmacology*
  • RNA, Messenger / antagonists & inhibitors
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / antagonists & inhibitors
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Structure-Activity Relationship
  • Transcription, Genetic

Substances

  • Imidazoles
  • Protein Kinase Inhibitors
  • Pyrimidines
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Clk dual-specificity kinases
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases