C3G dynamically associates with nuclear speckles and regulates mRNA splicing

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Crk SH3 domain binding guanine nucleotide releasing factor (C3G) is crucial for mammalian embryonic development and exhibits nucleocytoplasmic exchange.
• Nuclear speckles are dynamic structures involved in RNA processing and splicing.

Purpose of the Study:

• To investigate the localization and function of C3G within the nucleus.
• To determine the role of C3G and its substrate Rap1 in nuclear speckles and RNA splicing.

Main Methods:

• Immunofluorescence microscopy to observe C3G and SC35 localization in nuclear speckles.
• CRISPR/Cas9-mediated knockdown and knockout to assess C3G's effect on splicing.
• Expression of kinase Clk1 and Rap1GAP to study C3G's regulation.

Main Results:

• C3G localizes to SC35-positive nuclear speckles and its association is reversible with transcription/splicing inhibition.
• C3G's nuclear speckle localization depends on the actin cytoskeleton and is affected by Clk1 kinase.
• Rap1, a C3G substrate, is also found in nuclear speckles, and its signaling impacts speckle morphology.
• C3G knockdown/knockout alters splicing activity of artificial and endogenous genes (CD44) and reduces splicing factor levels.

Conclusions:

• C3G and Rap1 are identified as novel components of nuclear speckles.
• C3G plays a significant role in regulating cellular RNA splicing activity.