SAP155-mediated splicing of FUSE-binding protein-interacting repressor serves as a molecular switch for c-myc gene expression.

Abstract

The Far UpStream Element (FUSE)-binding protein-interacting repressor (FIR), a c-myc transcriptional suppressor, is alternatively spliced removing the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. SAP155 is a subunit of the essential splicing factor 3b (SF3b) subcomplex in the spliceosome. This study aims to study the significance of the FIR-SAP155 interaction for the coordination of c-myc transcription, pre-mRNA splicing, and c-Myc protein modification, as well as to interrogate FIRΔexon2 for other functions relating to altered FIR pre-mRNA splicing. Knockdown of SAP155 or FIR was used to investigate their reciprocal influence on each other and on c-myc transcription, pre-mRNA splicing, and protein expression. Pull down from HeLa cell nuclear extracts revealed the association of FIR, FIRΔexon2, and SF3b subunits. FIR and FIRΔexon2 were coimmunoprecipitated with SAP155. FIR and FIRΔexon2 adenovirus vector (Ad-FIR and Ad-FIRΔexon2, respectively) were prepared to test for their influence on c-myc expression. FIR, SAP155, SAP130, and c-myc were coordinately upregulated in human colorectal cancer. These results reveal that SAP155 and FIR/FIRΔexon2 form a complex and are mutually upregulating. Ad-FIRΔexon2 antagonized Ad-FIR transcriptional repression of c-myc in HeLa cells. Because FIRΔexon2 still carries RRM1 and RRM2 and binding activity to FUSE, it is able to displace repression competent FIR from FUSE in electrophoretic mobility shift assays, thus thwarting FIR-mediated transcriptional repression by FUSE. Thus aberrant FIRΔexon2 production in turn sustained c-Myc expression. In conclusion, altered FIR and c-myc pre-mRNA splicing, in addition to c-Myc expression by augmented FIR/FIRΔexon2-SAP155 complex, potentially contribute to colorectal cancer development.

• Authors: Kazuyuki Matsushita, Toshiko Kajiwara, Mai Tamura, Mamoru Satoh, Nobuko Tanaka, Takeshi Tomonaga, Hisahiro Matsubara, Hideaki Shimada, Rei Yoshimoto, Akihiro Ito, Shuji Kubo, Tohru Natsume, David Levens, Minoru Yoshida, Fumio Nomura
• Journal: Molecular cancer research : MCR (Mol Cancer Res) Vol. 10 Issue 6 Pg. 787-99 (Jun 2012) ISSN: 1557-3125 [Electronic] United States
• PMID: 22496461 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: 2012 AACR

Chemical References

• MYC protein, human
• Phosphoproteins
• Proto-Oncogene Proteins c-myc
• RNA Precursors
• RNA Splicing Factors
• RNA-Binding Proteins
• Repressor Proteins
• Ribonucleoprotein, U2 Small Nuclear
• SF3B1 protein, human
• poly-U binding splicing factor 60KDa

Topics

• Alternative Splicing
• Amino Acid Sequence
• Base Sequence
• Blotting, Western
• Colorectal Neoplasms (genetics, metabolism, pathology)
• Exons (genetics)
• HCT116 Cells
• HEK293 Cells
• HeLa Cells
• Humans
• Introns (genetics)
• Molecular Sequence Data
• Mutation
• Phosphoproteins (genetics, metabolism)
• Protein Binding
• Proto-Oncogene Proteins c-myc (genetics, metabolism)
• RNA Interference
• RNA Precursors (genetics, metabolism)
• RNA Splicing Factors
• RNA-Binding Proteins (genetics, metabolism)
• Repressor Proteins (genetics, metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Ribonucleoprotein, U2 Small Nuclear (genetics, metabolism)
• Transcription, Genetic