BioID proximity mapping reveals novel SAP18 interactions in the prespliceosomal complex

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein Interactions

Background:

• SAP18 protein is known to associate with the SIN3 transcriptional repressor complex.
• SAP18 is also a component of the apoptosis- and splicing-associated protein (ASAP) complex, indicating a dual role.
• Understanding SAP18's in vivo interactions is crucial for elucidating its functions in gene regulation and splicing.

Purpose of the Study:

• To define the in vivo interactome of SAP18 using proximity-dependent biotin identification (BioID).
• To identify novel protein partners of SAP18 involved in transcriptional and splicing regulation.
• To investigate the role of SAP18 in the prespliceosome and its association with the ASAP complex.

Main Methods:

• Proximity-dependent biotin identification (BioID) followed by mass spectrometry.
• Biochemical fractionation and streptavidin purification of biotinylated proteins.
• Immunoprecipitation assays and mutational analysis of SAP18.

Main Results:

• Identification of new SIN3-associated interactors, including RBBP4 and SAP30BP.
• Discovery of 72 highly enriched spliceosomal proteins interacting with SAP18.
• Validation of novel interactions between SAP18 and prespliceosomal components (SNRNP70, SNRPA, SF3B1, U2AF1, SRSF1).
• Demonstration that a C-terminal SAP18 mutant impairs interaction with prespliceosomal proteins.

Conclusions:

• SAP18 possesses a complex interactome, linking it to both transcriptional repression and RNA splicing.
• SAP18 directly interacts with key components of the prespliceosome.
• The ASAP complex is involved in mediating SAP18's interaction with the prespliceosome.