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.