C3G dynamically associates with nuclear speckles and regulates mRNA splicing

Mol Biol Cell. 2018 May 1;29(9):1111-1124. doi: 10.1091/mbc.E17-07-0442.

Abstract

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Cell Line
  • Cell Line, Tumor
  • Cell Nucleus Structures / physiology
  • Guanine Nucleotide Exchange Factors / metabolism
  • Guanine Nucleotide-Releasing Factor 2 / metabolism*
  • Guanine Nucleotide-Releasing Factor 2 / physiology*
  • Humans
  • Nuclear Proteins
  • Protein Binding
  • Protein Serine-Threonine Kinases
  • Protein-Tyrosine Kinases
  • RNA Processing, Post-Transcriptional / physiology
  • RNA Splicing / physiology*
  • RNA, Messenger / metabolism
  • Serine-Arginine Splicing Factors / metabolism
  • Serine-Arginine Splicing Factors / physiology
  • Shelterin Complex
  • Signal Transduction
  • Spliceosomes
  • Telomere-Binding Proteins / metabolism

Substances

  • Guanine Nucleotide Exchange Factors
  • Guanine Nucleotide-Releasing Factor 2
  • Nuclear Proteins
  • RNA, Messenger
  • Shelterin Complex
  • TERF2IP protein, human
  • Telomere-Binding Proteins
  • SRSF2 protein, human
  • Serine-Arginine Splicing Factors
  • Clk dual-specificity kinases
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases